Method for identifying HIV-1 protease inhibitors with reduced metabolic affects through detection of human resistin polymorphisms

ABSTRACT

The invention provides a novel in vitro method for identifying HIV-1 protease inhibitors with reduced potential for inducing metabolic abnormalities. The invention further provides diagnostic methods for identifying patients who may be at risk of developing metabolic abnormalities subsequent to the administration of an HIV-1 protease inhibitor. The invention also provides novel polynucleotides associated with the incidence of HIV-1 protease inhibitor induced metabolic abnormalities. The invention also provides polynucleotide fragments corresponding to the genomic and/or coding regions of these polynucleotides which comprise at least one polymorphic locus per fragment. Allele-specific primers and probes which hybridize to these regions, and/or which comprise at least one polymorphic locus are also provided. The polynucleotides, primers, and probes of the present invention are useful in phenotype correlations, medicine, and genetic analysis. The invention further relates to diagnostic methods for using these novel polynucleotides in the diagnosis, treatment, and/or prevention of HIV-1 protease inhibitor induced metabolic abnormalities.

This application claims benefit to provisional application U.S. Ser. No.60/817,846 filed Jun. 30, 2006, under 35 U.S.C. 119(e). The entireteachings of the referenced application are incorporated herein byreference.

FIELD OF THE INVENTION

The invention provides a novel in vitro method for identifying HIV-1protease inhibitors with reduced potential for inducing metabolicabnormalities. The invention further provides diagnostic methods foridentifying patients who may be at risk of developing metabolicabnormalities subsequent to the administration of an HIV-1 proteaseinhibitor. The invention also provides novel polynucleotides associatedwith the incidence of HIV-1 protease inhibitor induced metabolicabnormalities. The invention also provides polynucleotide fragmentscorresponding to the genomic and/or coding regions of thesepolynucleotides which comprise at least one polymorphic locus perfragment. Allele-specific primers and probes which hybridize to theseregions, and/or which comprise at least one polymorphic locus are alsoprovided. The polynucleotides, primers, and probes of the presentinvention are useful in phenotype correlations, medicine, and geneticanalysis. The invention further relates to diagnostic methods for usingthese novel polynucleotides in the diagnosis, treatment, and/orprevention of HIV-1 protease inhibitor induced metabolic abnormalities.

BACKGROUND OF THE INVENTION

Highly active antiretroviral therapy (HAART) involves the use of acombination of three different classes of antiviral compounds for thetreatment of HIV infection. These compounds include the nucleosidereverse transcriptase inhibitors, non-nucleoside reverse transcriptaseinhibitors and protease inhibitors.

HAART has improved the outcomes in HIV-1 infected patients, however, itsuse is complicated by metabolic adverse events that are collectivelytermed HAART-associated lipodystrophy Carr, A., Aids, 17 Suppl 1: p.S141-8 (2003). These metabolic changes include fat atrophy/hypertrophy,dyslipidemia and insulin resistance. Although the mechanistic basisunderlying lipodystrophy associated with HAART is poorly understood, theassociated metabolic abnormalities are well established cardiovascularrisk factors (Grinspoon, S. et al., N Engl J Med, 352(1):48-62 (2005)).

Genetic association studies performed by the inventors revealed that asingle nucleotide polymorphism in the resistin gene provided evidence ofan association with a risk of developing lipodystrophy in patientsenrolled on HAART. Resistin was originally cloned as an adipocytokinethat provides a mechanistic link between obesity and insulin resistance(Steppan, C. M., et al., Nature, 409(6818):307-12 (2001)). In mice,hyperresistinemia results in insulin resistance (Rajala, M. W., et al.,J Clin Invest, 111(2):225-30 (2003)) and knockout studies have shownthat resistin deficient mice are protected from insulin resistance(Banerjee, R. R., et al., Science, 303(5661):1195-8 (2004)). In humans,resistin has been shown to be associated with components oflipodystrophy syndrome in some (Conneely, K. N., et al., Diabetologia,47(10): 1782-8 (2004), Mattevi, V. S., et al., Hum Genet, 115(3):208-12(2004)), but not all studies (Ochi, M., et al., Diabetes Res Clin Pract,61(3): p. 191-8 (2003)).

Aspartyl proteases comprise a group of proteolytic enzymes that sharethe same catalytic mechanism (Dash, C., et al., Crit Rev Biochem MolBiol, 38(2):89-119 (2003)). Members of this family include HIV-1protease and at least five human enzymes. In pre-clinical development,the human aspartyl proteases are used to counter-screen HIV-1 proteaseinhibitors for selectivity toward HIV-1 protease. However, selectivitytoward HIV-1 protease can vary widely across HIV-1 protease inhibitors(Ohtaka, H., et al., Int J Biochem Cell Biol, 36(9):1787-99 (2004)).

The present invention provides, for the first time, an in vitro assayfor identifying protease inhibitors that either lack or have a lowerpropensity for causing metabolic abnormalities in patients.

The present invention also provides genetic polymorphisms in theresistin gene which may cause alterations in the function and/orexpression of resistin. Such polymorphisms may genetically predisposecertain individuals to an increased risk of developing metabolicabnormalities, particularly in response to HIV protease-inhibitortherapy. Genotypes of such polymorphisms may predict each individual'ssusceptibility to metabolic abnormalities, and thus will be useful inidentifying a group of patients that may be subject to modified HAARTtreatment regimens. Alternatively, the identification of such a groupmay preclude one or more individuals within said group from beingadministered HAART therapy.

SUMMARY OF THE INVENTION

The present invention also relates to a method for screening for HIV-1protease inhibitor compounds with a diminished ability to increase anindividuals likelihood of developing HIV-1 protease inhibitor-dependentmetabolic abnormalities upon the administration of the same. In specificembodiments of the invention, such a method comprises the step ofcombining a mixture of resistin and cathepsin D or E with either one ormore test drugs, compounds, or other therapeutic agents, or a controlcompound as shown in FIG. 2; followed by the step of measuring the levelof proteolyzed resistin between the test and control samples, andselecting the test compound with a diminished ability to inhibitcathepsin D- or cathepsin E-dependent degradation of resistin relativeto a control compound.

The present invention relates to methods of predicting whether a patienthas an increased risk of developing HIV-1 protease inhibitor-dependentmetabolic abnormalities upon the administration of a pharmaceuticallyacceptable level of a HIV-1 protease inhibitor; whether said patientrequires a lower level of administered HIV-1 protease inhibitor to limitthe risk of developing said HIV-1 protease inhibitor-dependent metabolicabnormalities; or whether said patient may be administered a higherlevel of administered HIV-1 protease inhibitor without the risk or of alower risk of developing said HIV-1 protease inhibitor-dependentmetabolic abnormalities, comprising the step of assessing the level ofresistin expression or resistin plasma/serum levels resulting from theadministration of a HIV-1 protease inhibitor relative to a controlcompound, wherein an elevated level of resistin expression or resistinplasma/serum levels is indicative of an increased risk of developingHIV-1 protease inhibitor-dependent metabolic abnormalities subsequent tothe administration of a HIV-1 protease inhibitor.

The invention relates to a nucleic acid molecule which comprises, oralternatively consists of, at least one single nucleotide polymorphismwithin the resistin genomic sequence at a specific polymorphic locus. Ina particular embodiment the invention relates to the variant allele ofthe resistin gene or polynucleotide having at least one singlenucleotide polymorphism, which variant allele differs from a referenceallele by one nucleotide at the site(s) identified in FIGS. 3A-B, and/orFIGS. 4A-B, or elsewhere herein. The complementary sequence of each ofthese nucleic acid molecules are also provided. The nucleic acidmolecules can be comprised of DNA or RNA, can be double- orsingle-stranded, and may comprise fragments. Fragments can be, forexample, about 5 to about 10, about 5 to about 15, about 10 to about 20,about 15 to about 25, about 10 to about 30, about 10 to about 50, orabout 10 to about 100 bases long, and preferably comprise at least onepolymorphic allele.

In another embodiment, the invention relates to the reference or wildtype allele of a gene or polynucleotide having a polymorphic locus, inwhich said reference or wild type allele differs from a variant alleleby one nucleotide at the polymorphic site(s) identified in FIGS. 3A-B,and/or FIGS. 4A-B, or elsewhere herein. The complementary sequence ofeach of these nucleic acid molecules are also provided. The nucleic acidmolecules can be comprised of DNA or RNA, can be double- orsingle-stranded, and may comprise fragments. Fragments can be, forexample, about 5 to about 10, about 5 to about 15, about 10 to about 20,about 15 to about 25, about 10 to about 30, about 10 to about 50, orabout 10 to about 100 bases long, and preferably comprise at least onepolymorphic locus.

The invention further provides variant and reference allele-specificoligonucleotides that hybridize to a nucleic acid molecule comprising atleast one polymorphic locus, in addition to the complement of saidoligonucleotide. These oligonucleotides can be probes or primers.

The invention further provides oligonucleotides that may be used toamplify a portion of either the variant or reference sequencescomprising at least one polymorphic locus of the present invention, inaddition to providing oligonucleotides that may be used to sequence saidamplified sequence. The invention further provides a method of analyzinga nucleic acid from a DNA sample using said amplification and sequencingprimers to assess whether said sample contains the reference or variantnucleotide (allele) at the polymorphic locus, comprising the steps ofamplifying a sequence using appropriate oligonucleotide primers foramplifying across a polymorphic locus, and sequencing the resultingamplified product using appropriate sequencing primers to sequence saidproduct to determine whether the variant or reference base is present atthe polymorphic locus.

The invention further provides a method of analyzing a nucleic acid frompatient sample(s) using said amplification and sequencing primers toassess whether said sample(s) contain the reference or variantnucleotide (allele) at the polymorphic locus in an effort to identifypopulations at risk of developing HIV-1 protease inhibitor-dependentmetabolic abnormalities upon administration of a HIV-1 proteaseinhibitor, comprising the steps of amplifying a sequence usingappropriate oligonucleotide primers for amplifying across a polymorphiclocus, and sequencing the resulting amplified product using appropriatesequencing primers to sequence said product to determine whether thevariant or reference nucleotide is present at the polymorphic locus.

The invention further provides oligonucleotides that may be used togenotype patient sample(s) to assess whether said sample(s) contain thereference or variant nucleotide (allele) at the polymorphic site(s). Theinvention provide a method of using oligonucleotides that may be used togenotype a patient sample to assess whether said sample contains thereference or variant nucleotide (allele) at the polymorphic locus. Anembodiment of the method comprises the steps of amplifying a sequenceusing appropriate oligonucleotide primers for amplifying across apolymorphic locus, and subjecting the product of said amplification to agenetic bit analysis (GBA) reaction.

The invention provides a method of using oligonucleotides that may beused to genotype patient sample(s) to identify individual(s) at risk ofdeveloping HIV-1 protease inhibitor-dependent metabolic abnormalitiesupon administration of a HIV-1 protease inhibitor to assess whether saidsample(s) contains the reference or variant nucleotide (allele) at oneor more polymorphic loci. An embodiment of the method comprises thesteps of amplifying a sequence using appropriate oligonucleotide primersfor amplifying across a polymorphic locus, and subjecting the product ofsaid amplification to a genetic bit analysis (GBA) reaction, andoptionally determining the statistical association between either thereference or variant allele at the polymorphic site(s) to the incidenceof HIV-1 protease inhibitor-dependent metabolic abnormalities.

The invention provides a method of using oligonucleotides that may beused to genotype patient sample(s) to identify ethnic population(s) thatmay be at risk of developing HIV-1 protease inhibitor-dependentmetabolic abnormalities upon administration of a HIV-1 proteaseinhibitor to assess whether said sample(s) contains the reference orvariant nucleotide (allele) at one or more polymorphic loci comprisingthe steps of amplifying a sequence using appropriate oligonucleotideprimers for amplifying across a polymorphic locus, and subjecting theproduct of said amplification to a genetic bit analysis (GBA) reaction,and optionally determining the statistical association between eitherthe reference or variant allele at the polymorphic site(s) to theincidence of HIV-1 protease inhibitor-dependent metabolic abnormalities.

The invention further provides a method of analyzing a nucleic acid fromone or more individuals. The method allows the determination of whetherthe reference or variant base is present at any one, or more, of thepolymorphic sites identified FIGS. 3A-B, and/or FIGS. 4A-B, or elsewhereherein. Optionally, a set of nucleotides occupying a set of thepolymorphic loci shown in FIGS. 3A-B, and/or FIGS. 4A-B, or elsewhereherein, is determined. This type of analysis can be performed on anumber of individuals, who are also tested (previously, concurrently orsubsequently) for the presence of HIV-1 protease inhibitor-dependentmetabolic abnormalities. The presence or absence of a HIV-1 proteaseinhibitor-dependent metabolic abnormalities phenotype is then correlatedwith said nucleotide or set of nucleotides present at the polymorphiclocus or loci in the individuals tested.

The invention further relates to a method of predicting the presence,absence, likelihood of the presence or absence, or severity of a HIV-1protease inhibitor-dependent metabolic abnormalities or related disorderassociated with a particular genotype. The method comprises obtaining anucleic acid sample from an individual and determining the identity ofone or more nucleotides at specific polymorphic loci of nucleic acidmolecules described herein, wherein the presence of a particular base atthat site is correlated with the incidence of HIV-1 proteaseinhibitor-dependent metabolic abnormalities or related disorder, therebypredicting the presence, absence, likelihood of the presence or absence,or severity, of the HIV-1 protease inhibitor-dependent metabolicabnormalities phenotype or related disorder in the individual.

The invention further relates to polynucleotides having one or morepolymorphic loci comprising one or more variant alleles. The inventionalso relates to said polynucleotides lacking a start codon. Theinvention further relates to polynucleotides of the present inventioncontaining one or more variant alleles wherein said polynucleotidesencode a polypeptide of the present invention. The invention relates topolypeptides of the present invention containing one or more variantamino acids encoded by one or more variant alleles.

The present invention relates to antisense oligonucleotides capable ofhybridizing to the polynucleotides of the present invention. Preferably,such antisense oligonucleotides are capable of discriminating betweenthe reference or variant allele of the polynucleotide, preferably at oneor more polymorphic sites of said polynucleotide.

The present invention relates to siRNA or RNAi oligonucleotides capableof hybridizing to the polynucleotides of the present invention.Preferably, such siRNA or RNAi oligonucleotides are capable ofdiscriminating between the reference or variant allele of thepolynucleotide, preferably at one or more polymorphic sites of saidpolynucleotide.

The present invention also relates to zinc finger proteins capable ofbinding to the polynucleotides of the present invention. Preferably,such zinc finger proteins are capable of discriminating between thereference or variant allele of the polynucleotide, preferably at one ormore polymorphic sites of said polynucleotide.

The present invention also relates to recombinant vectors, which includethe isolated nucleic acid molecules of the present invention, and tohost cells containing the recombinant vectors, as well as to methods ofmaking such vectors and host cells, in addition to their use in theproduction of polypeptides or peptides provided herein using recombinanttechniques. Synthetic methods for producing the polypeptides andpolynucleotides of the present invention are provided. Also provided arediagnostic methods for detecting diseases, disorders, and/or conditionsrelated to the polypeptides and polynucleotides provided herein, andtherapeutic methods for treating such diseases, disorders, and/orconditions. The invention further relates to screening methods foridentifying binding partners of the polypeptides.

The invention relates to a method of analyzing at least one nucleic acidsample, comprising the step of determining the nucleic acid sequencefrom one or more samples at one or more polymorphic loci in the humanresistin gene, wherein the presence of the reference allele at said oneor more polymorphic loci is indicative of an increased risk ofdeveloping HIV-1 protease inhibitor-dependent metabolic abnormalities ina patient receiving HIV-1 protease inhibitor therapy.

The invention relates to a method of analyzing at least one nucleic acidsample, comprising the step of determining the nucleic acid sequencefrom one or more samples at one or more polymorphic loci in the humanresistin gene, wherein the presence of the variant allele at said one ormore polymorphic loci is indicative of a decreased risk of developingHIV-1 protease inhibitor-dependent metabolic abnormalities in a patientreceiving HIV-1 protease inhibitor therapy.

The invention further relates to a method of constructing haplotypesusing the isolated nucleic acids referred to in FIGS. 3A-B and/or FIGS.4A-B, or elsewhere herein, comprising the step of grouping at least twosaid nucleic acids.

The invention further relates to a method of constructing haplotypesfurther comprising the step of using said haplotypes to identify anindividual for the presence of HIV-1 protease inhibitor-dependentmetabolic abnormalities or related disease phenotype, and correlatingthe presence of the disease phenotype with said haplotype.

The invention further relates to a library of nucleic acids, each ofwhich comprises one or more polymorphic positions within a gene encodingthe human resistin protein, wherein said polymorphic positions areselected from a group consisting of the polymorphic positions providedin FIGS. 3A-B and FIGS. 4A-B.

The invention further relates to a library of nucleic acids, wherein thesequence at said aforementioned polymorphic position is selected fromthe group consisting of the polymorphic position identified in FIGS.3A-B, and/or FIGS. 4A-B, or elsewhere herein, the complimentary sequenceof said sequences, and/or fragments of said sequences.

The invention further relates to a kit for identifying an individual atrisk of developing HIV-1 protease inhibitor-dependent metabolicabnormalities or related disorder upon administration of apharmaceutically acceptable amount of a HIV-1 protease inhibitor,wherein said kit comprises oligonucleotide primers capable ofidentifying the nucleotide residing at one or more polymorphic loci ofthe human resistin gene, wherein the presence of the variant allele atsaid one or more polymorphic loci is indicative of a decreased risk ofdeveloping HIV-1 protease inhibitor-dependent metabolic abnormalities ina patient receiving HIV-1 protease inhibitor therapy, while the presenceof the reference allele at said one or more polymorphic loci isindicative of an increased risk of developing HIV-1 proteaseinhibitor-dependent metabolic abnormalities in a patient receiving HIV-1protease inhibitor therapy.

The invention further relates to a kit for identifying an individual atrisk of developing HIV-1 protease inhibitor-dependent metabolicabnormalities or related disorder upon administration of apharmaceutically acceptable amount of a HIV-1 protease inhibitor,wherein said kit comprises oligonucleotide primers capable ofidentifying the nucleotide residing at one or more polymorphic loci ofthe human resistin gene, wherein the presence of the variant allele atsaid one or more polymorphic loci is indicative of a decreased risk ofdeveloping HIV-1 protease inhibitor-dependent metabolic abnormalities ina patient receiving HIV-1 protease inhibitor therapy, while the presenceof the reference allele at said one or more polymorphic loci isindicative of an increased risk of developing HIV-1 proteaseinhibitor-dependent metabolic abnormalities in a patient receiving HIV-1protease inhibitor therapy, and wherein said oligonucleotides hybridizeimmediately adjacent to said one or more polymorphic positions, orwherein said primer(s) hybridizes to said polymorphic positions suchthat the central position of the primer aligns with the polymorphicposition of said gene.

The invention further relates to a method for predicting the likelihoodthat an individual will be diagnosed as being at risk of developingHIV-1 protease inhibitor-dependent metabolic abnormalities or relateddisorder upon administration of a pharmaceutically acceptable amount ofa HIV-1 protease inhibitor comprising the step of determining thenucleotide present within at least one or more nucleic acid sample(s)from an individual to be assessed at one or more polymorphic position(s)of the human resistin gene sequence selected from the group consistingof: SEQ ID NOS:1 and/or 2, wherein the presence of the referencenucleotide at the one or more polymorphic position(s) indicates that theindividual has an increased likelihood of being diagnosed as at risk ofdeveloping HIV-1 protease inhibitor-dependent metabolic abnormalities orrelated disorder upon administration of a pharmaceutically acceptableamount of an HIV-1 protease inhibitor as compared to an individualhaving the variant allele at said polymorphic position(s).

The invention further relates to a method for predicting the likelihoodthat an individual will be diagnosed as being at risk of developing aHIV-1 protease inhibitor-dependent metabolic abnormalities or relateddisorder upon administration of a pharmaceutically acceptable amount ofa HIV-1 protease inhibitor comprising the step of determining thenucleotide present within at least one or more nucleic acid sample(s)from an individual to be assessed at one or more polymorphic position(s)of the human resistin gene sequence selected from the group consistingof: SEQ ID NOS:1 and/or 2, wherein the presence of the variantnucleotide at the one or more polymorphic position(s) indicates that theindividual has an decreased likelihood of being diagnosed as at risk ofdeveloping HIV-1 protease inhibitor-dependent metabolic abnormalities orrelated disorder upon administration of a pharmaceutically acceptableamount of an HIV-1 protease inhibitor as compared to an individualhaving the reference allele at said polymorphic position(s).

The invention further relates to a method for predicting the likelihoodthat an individual will be diagnosed as being at risk of developingHIV-1 protease inhibitor-dependent metabolic abnormalities or relateddisorder upon administration of a pharmaceutically acceptable amount ofa HIV-1 protease inhibitor comprising the step of determining thenucleotide present within at least one or more nucleic acid sample(s)from an individual to be assessed at one or more polymorphic position(s)of the human resistin gene sequence selected from the group consistingof: nucleotide position 1398 of SEQ ID NOS:1 or 2, wherein the presenceof the reference nucleotide at the one or more polymorphic position(s)indicates that the individual has an increased likelihood of beingdiagnosed as at risk of developing HIV-1 protease inhibitor-dependentmetabolic abnormalities or related disorder upon administration of apharmaceutically acceptable amount of a HIV-1 protease inhibitor ascompared to an individual having the variant allele at said polymorphicposition(s).

The invention further relates to a method for predicting the likelihoodthat an individual will be diagnosed as being at risk of developing aHIV-1 protease inhibitor-dependent metabolic abnormalities or relateddisorder upon administration of a pharmaceutically acceptable amount ofa HIV-1 protease inhibitor comprising the step of determining thenucleotide present within at least one or more nucleic acid sample(s)from an individual to be assessed at one or more polymorphic position(s)of the human resistin gene sequence selected from the group consistingof: nucleotide position 1398 of SEQ ID NOS:1 or 2, wherein the presenceof the variant nucleotide at the one or more polymorphic position(s)indicates that the individual has a decreased likelihood of beingdiagnosed as at risk of developing HIV-1 protease inhibitor-dependentmetabolic abnormalities or related disorder upon administration of apharmaceutically acceptable amount of a HIV-1 protease inhibitor ascompared to an individual having the reference allele at saidpolymorphic position(s).

A method of decreasing the risk of a patient developing HIV-1 proteaseinhibitor-dependent metabolic abnormalities upon administration of anHIV-1 protease inhibitor comprising the step of administering either anagent that inhibits resistin activity, or an agent that decreases theexpression level of resistin in a patient, wherein said agent is a PPARγagonist, including but not limited to either rosiglitazone orpioglitazone.

A method of decreasing the level of HIV-1 protease inhibitor-dependentmetabolic abnormalities in a patient comprising the step ofadministering either an agent that inhibits resistin activity, or anagent that decreases the expression level of resistin in a patient inresponse to or in combination with the administration of an HIV-1protease inhibitor, wherein said agent is a PPARγ agonist, including butnot limited to either rosiglitazone or pioglitazone, or bothrosiglitazone or pioglitazone.

A method of identifying HIV patients who may benefit from theadministration of a pharmaceutically acceptable amount of atazanavir,comprising the step of comparing the plasma/serum level of resistin in apatient sample relative to a reference sample, wherein an elevated levelof resistin relative to the reference sample is indicative of a patientwho may have an increased risk of developing HIV-1 proteaseinhibitor-dependent metabolic abnormalities and who would benefit fromthe administration of a pharmaceutically acceptable amount ofatazanavir.

BRIEF DESCRIPTION OF THE FIGURES/DRAWINGS

FIG. 1 shows that resistin is cleaved by the aspartyl proteases HIV-1protease, Cathepsin D and Cathepsin E. In each reaction, 1 υg ofalkylated and reduced resistin was co-incubated, for 24 hr with 1 υgHIV-1 protease, 0.1 υg Cathepsin D, 0.1 υg Cathepsin E, 0.1 υg trypsinor 0.1 υg renin. Control experiments were also performed, in parallel,in which either protease or resistin was omitted from the reaction,respectively. Protease digestion products were separated by 4-12%SDS-PAGE, followed by coomassie blue staining. Abbreviations are asfollows: resistin (“Retn”), HIV-1 protease (“HIV-1 PR”); cathepsin D(“CathD”); cathepsin E (“CathE”); trypsin (“Trp”); and renin (“Ren”).Arrows indicate resistin cleavage products.

FIG. 2 shows an evaluation of HIV-1 protease inhibitors on cleavage ofresistin by proteases. Protease digestion experiments were performed asin FIG. 1 with the addition of atazanavir (“ATV”), ritonavir (“RTV”) orlopinovir (“LPV”) at the respective concentrations indicated.

FIGS. 3A-B show the polynucleotide sequence (SEQ ID NO:1) of SNP1 alleleC of the human resistin genomic sequence comprising, or alternativelyconsisting of, a predicted polynucleotide polymorphic locus located atnucleotide 1398 of SEQ ID NO:1. The polynucleotide sequence contains asequence of 2369 nucleotides. The nucleotide at the polymorphic locuswithin this allele is a “C” and is denoted in bold and doubleunderlining. Exons encoding the resistin polypeptide are denoted bysingle underlining, while non-underlined sequence represent introns.

FIGS. 4A-B show the polynucleotide sequence (SEQ ID NO:2) of SNP1 alleleT of the human resistin genomic sequence comprising, or alternativelyconsisting of, a predicted polynucleotide polymorphic locus located atnucleotide 1398 of SEQ ID NO:2. The polynucleotide sequence contains asequence of 2369 nucleotides. The nucleotide at the polymorphic locuswithin this allele is a “T” and is denoted in bold and doubleunderlining. Exons encoding the resistin polypeptide are denoted bysingle underlining, while non-underlined sequence represents introns.

FIG. 5 shows the statistical association between human resistin SNP1alleles “C” and “T” with the incidence of HIV-1 proteaseinhibitor-dependent metabolic abnormalities in patients administeredHAART therapy. Results are shown in terms of fold incidence of eachgenotype residing in a patient that was part of the high-risk clusterfor HIV-1 protease inhibitor-dependent metabolic abnormalities. Asshown, “T” allele homozygous patients (“T/T”) at the SNP1 locus have thehighest incidence of HIV-1 protease inhibitor-dependent metabolicabnormalities; heterozygous patients (“C/T”) at the SNP1 locus have alower incidence of HIV-1 protease inhibitor-dependent metabolicabnormalities compared to homozygous “T/T” allele patients; while “C”allele homozygous patients (“C/C”) at the SNP1 locus have asignificantly lower incidence of HIV-1 protease inhibitor-dependentmetabolic abnormalities compared to homozygous “T” and heterozygous(“C/T”) allele patients.

Table I provides a summary of the SNPs of the present invention.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a method of screening for HIV-1protease inhibitor compounds with diminished ability to increase anindividuals likelihood of developing HIV-1 protease inhibitor-dependentmetabolic abnormalities upon the administration of the same. The presentinvention is based on the novel discovery that resistin is sensitive toproteolytic degradation by aspartyl proteases cathepsin D and E as shownin FIG. 1. Since cathespin D and E are inhibited by some HIV-1 proteaseinhibitors, inhibition of cathespin D and E can result in increasedlevels of resistin and thus result in metabolic abnormalities. Themethod of the present invention comprises the step of incubating amixture of resistin and cathepsin D or E with either one or more testdrugs, compounds, or therapeutic agents, in addition to a controlcompound as shown in FIG. 2; followed by the step of measuring the levelof proteolyzed resistin between the test and control samples, andselecting the test compound with a diminished ability to inhibitcathepsin D- or cathepsin E-dependent degradation of resistin relativeto a control compound. Such an HIV-1 protease inhibitor compound wouldbe expected to have a diminished ability to cause metabolicabnormalities in a patient.

The present invention also relates to a nucleic acid molecule comprisinga single nucleotide polymorphism (SNP) at a specific location, referredto herein as the polymorphic locus, and complements thereof. The nucleicacid molecule, e.g., a gene, which includes the SNP has at least twoalleles, referred to herein as the reference allele and the variantallele. The reference allele (prototypical or wild type allele)typically corresponds to the nucleotide sequence of the native form ofthe nucleic acid molecule.

The present invention pertains to novel polynucleotides of the humanresistin gene comprising at least one single nucleotide polymorphism(SNP) which has been shown to be associated with the incidence of HIV-1protease inhibitor-dependent metabolic abnormalities in patientsadministered HIV-1 protease inhibitors. These resistin SNPs wereidentified by sequencing the resistin genomic sequence of a large numberof individuals that were subjected to HIV-1 protease inhibitor therapy,and comparing the resistin sequences of those individuals who developedHIV-1 protease inhibitor-dependent metabolic abnormalities to thoseindividuals who did not develop HIV-1 protease inhibitor-dependentmetabolic abnormalities. Each of the novel resistin SNPs were located innon-coding regions of the resistin gene and are thought to affect theexpression levels of resistin in those patients containing one or moreof these SNPs.

The present invention also relates to variant alleles of the describedgene and to complements of the variant alleles. The variant allelediffers from the reference allele by one nucleotide at the polymorphiclocus identified in the FIGS. 3A-B, and/or FIGS. 4A-B.

The invention further relates to fragments of the variant alleles andportions of complements of the variant alleles which comprise the siteof the SNP (e.g., polymorphic locus) and are at least 10 nucleotides inlength. Fragments can be, for example, about 5-10, about 5-15, about10-20, about 5-25, about 10-30, about 10-50 or about 10-100 bases long.For example, a portion of a variant allele which is about 10 nucleotidesin length comprises at least one single nucleotide polymorphism (thenucleotide which differs from the reference allele at the polymorphiclocus) and nine additional nucleotides which flank the site in thevariant allele. These additional nucleotides can be on one or both sidesof the polymorphism. Polymorphisms which are the subject of thisinvention are defined in FIGS. 3A-B, and/or FIGS. 4A-B herein.

Specifically, the invention relates to the human resistin gene having anucleotide sequence according to FIGS. 3A-B or FIGS. 4A-B (SEQ ID NOs:1or 2) comprising a single nucleotide polymorphism at a polymorphic locusat nucleotide 1398 of SEQ ID NOs:1 or 2. The reference nucleotide forthe polymorphic locus at nucleotide 1398 is “C”. The variant nucleotidefor the polymorphic locus at nucleotide 1398 is “T”. The nucleotidesequences of the present invention can be double- or single-stranded.

The invention further relates to a portion of the human resistin genecomprising one or more polymorphic loci selected from the groupconsisting of: nucleotide 1398 of SEQ ID NOs:1 or 2.

The single nucleotide polymorphisms described herein derive from theresistin gene that have been shown to be associated, for the first time,with the incidence of HIV-1 protease inhibitor-dependent metabolicabnormalities or related disorders. Specifically, the reference singlenucleotide polymorphisms of the human resistin gene described hereinhave been demonstrated to statistically increase an individualssusceptibility to developing HIV-1 protease inhibitor-dependentmetabolic abnormalities upon the administration of an increased dose ofa HIV-1 protease inhibitor.

The human resistin gene was chosen as a candidate gene to investigatethe potential of it comprising one or more single nucleotidepolymorphisms associated with HIV-1 protease inhibitor-dependentmetabolic abnormalities phenotype, and in particular, the potential ofidentifying a resistin SNP associated with the incidence of HIV-1protease inhibitor-dependent metabolic abnormalities phenotype upon theadministration of an HIV-1 protease inhibitor, based upon theassociation of resistin in glucose metabolism, obesity and insulinresistance.

The invention further provides allele-specific oligonucleotides thathybridize to the human resistin gene, or fragments or complementsthereof, comprising one or more single nucleotide polymorphisms and/orpolymorphic locus. Such oligonucleotides are expected to hybridize toone polymorphic allele of the nucleic acid molecules described hereinbut not to the other polymorphic allele(s) of the sequence. Thus, sucholigonucleotides can be used to determine the presence or absence ofparticular alleles of the polymorphic sequences described herein and todistinguish between reference and variant allele for each form. Theseoligonucleotides can be probes or primers.

The invention further provides a method of analyzing a nucleic acid froman individual to identify the presence or absence of a particularnucleotide at a given polymorphic locus and to distinguish between thereference and variant allele at each locus. The method determines whichbase is present at any one of the polymorphic loci shown in FIGS. 3A-Bor FIGS. 4A-B (SEQ ID NOs:1 or 2), or elsewhere herein. Optionally, aset of bases occupying a set of the polymorphic loci shown in FIGS. 3A-Bor FIGS. 4A-B (SEQ ID NOs:1 or 2) is determined. This type of analysiscan be performed on a number of individuals, who are also tested(previously, concurrently or subsequently) for the presence of HIV-1protease inhibitor-dependent metabolic abnormalities phenotype in thepresence or absence of a HIV-1 protease inhibitor. The presence orabsence of HIV-1 protease inhibitor-dependent metabolic abnormalitiesphenotype is then correlated with a base or set of bases present at thepolymorphic locus or loci in the patient and/or sample tested.

Thus, the invention further relates to a method of predicting thepresence, absence, likelihood of the presence or absence, or severity ofa particular HIV-1 protease inhibitor-dependent metabolic abnormalitiesphenotype associated with a particular genotype in the presence orabsence of an increased dose of a HIV-1 protease inhibitor. The methodcomprises obtaining a nucleic acid sample from an individual anddetermining the identity of one or more bases (nucleotides) at on ormore polymorphic loci of the nucleic acid molecules described herein,wherein the presence of a particular base is correlated with theincidence of HIV-1 protease inhibitor-dependent metabolic abnormalitiesphenotype or an increased risk of developing HIV-1 proteaseinhibitor-dependent metabolic abnormalities phenotype in the presence ofa HIV-1 protease inhibitor, thereby predicting the presence, absence,likelihood of the presence or absence, or severity of HIV-1 proteaseinhibitor-dependent metabolic abnormalities in the individual or sample.The correlation between a particular polymorphic form of a gene and aphenotype can thus be used in methods of diagnosis of that phenotype, aswell as in the development of treatments for the phenotype.

Definitions

An “oligonucleotide” can be DNA or RNA, and single- or double-stranded.An oligonucleotide may be used as either a “primer” or a “probe”.Oligonucleotides can be naturally occurring or synthetic, but aretypically prepared by synthetic means. An oligonucleotide primer, forexample, may be designed to hybridize to the complementary sequence ofeither the sense or antisense strand of a specific target sequence, andmay be used alone or as a pair, such as in DNA amplification reactions,and may or may not comprise one or more polymorphic loci of the presentinvention. An oligonucleotide probe may also be designed to hybridize tothe complementary sequence of either the sense or antisense strand of aspecific target sequence, and may be used alone or as a pair, such as inDNA amplification reactions, but necessarily will comprise one or morepolymorphic loci of the present invention. Preferred oligonucleotides ofthe invention include fragments of DNA, or their complements thereof, ofthe human resistin gene, and may comprise one or more of the polymorphicloci shown or described in FIGS. 3A-B, FIGS. 4A-B, or as describedelsewhere herein. The fragments can be between 10 and 250 bases, and, inspecific embodiments, are between about 5 to about 10, about 5 to about15, about 10 to about 20, about 15 to about 25, about 10 to about 30,about 10 to about 50, or about 10 to about 100 bases in length. Forexample, the fragment can be 40 bases in length. The polymorphic locuscan occur within any nucleotide position of the fragment, including ateither terminus or directly in the middle, for example. The fragmentscan be from any of the allelic forms of DNA shown or described herein.

As used herein, the terms “nucleotide”, “base” and “nucleic acid” areintended to be equivalent. The terms “nucleotide sequence”, “nucleicacid sequence”, “nucleic acid molecule” and “segment” are intended to beequivalent.

Hybridization probes are oligonucleotides which bind in a base-specificmanner to a complementary strand of nucleic acid and are designed toidentify the allele at one or more polymorphic loci within the resistingene of the present invention. Such probes include peptide nucleicacids, as described in Nielsen et al., Science 254, 1497-1500 (1991).Probes can be any length suitable for specific hybridization to thetarget nucleic acid sequence. The most appropriate length of the probemay vary depending upon the hybridization method in which it is beingused; for example, particular lengths may be more appropriate for use inmicrofabricated arrays, while other lengths may be more suitable for usein classical hybridization methods. Such optimizations are known to theskilled artisan. Suitable probes can range from about 12 nucleotides toabout 25 nucleotides in length. For example, probes and primers can beabout 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 25, 26, or about 40nucleotides in length. The probe preferably comprises at least onepolymorphic locus occupied by any of the possible variant nucleotides.For comparison purposes, the present invention also encompasses probesthat comprise the reference nucleotide at at least one polymorphiclocus. The nucleotide sequence can correspond to the coding sequence ofthe allele or to the complement of the coding sequence of the allele,where applicable.

As used herein, the term “primer” refers to a single-strandedoligonucleotide which acts as a point of initiation of template-directedDNA synthesis under appropriate conditions. Such DNA synthesis reactionsmay be carried out in the traditional method of including all fourdifferent nucleoside triphosphates (e.g., in the form ofphosphoramidates, for example) corresponding to adenine, guanine,cytosine and thymine or uracil nucleotides, and an agent forpolymerization, such as DNA or RNA polymerase or reverse transcriptasein an appropriate buffer and at a suitable temperature. Alternatively,such a DNA synthesis reaction may utilize only a single nucleoside(e.g., for single base-pair extension assays). The appropriate length ofa primer depends on the intended use of the primer, but typically rangesfrom about 10 to about 30 nucleotides. Short primer molecules generallyrequire cooler temperatures to form sufficiently stable hybrid complexeswith the template. A primer need not reflect the exact sequence of thetemplate, but must be sufficiently complementary to hybridize with atemplate. The term. “primer site” refers to the area of the target DNAto which a primer hybridizes. The term primer pair refers to a set ofprimers including a 5′ (upstream) primer that hybridizes with the 5′ endof the DNA sequence to be amplified and a 3′ (downstream) primer thathybridizes with the complement of the 3′ end of the sequence to beamplified.

As used herein, “linkage” describes the tendency of genes, alleles, locior genetic markers to be inherited together as a result of theirlocation on the same chromosome. It can be measured by percentrecombination between the two genes, alleles, loci or genetic markers.

As used herein, “polymorphism” refers to the occurrence of two or moregenetically determined alternative sequences or alleles in a population.A “polymorphic locus” is a marker or site at which divergence from areference allele occurs. The phrase “polymorphic loci” is meant to referto two or more markers or sites at which divergence from two or morereference alleles occurs. Preferred markers have at least two alleles,each occurring at frequency of greater than 1%, and more preferablygreater than 10% or 20% of a selected population. A polymorphic locusmay be as small as one base pair. Polymorphic loci include restrictionfragment length polymorphisms, variable number of tandem repeats(VNTR's), hypervariable regions, minisatellites, dinucleotide repeats,trinucleotide repeats, tetranucleotide repeats, simple sequence repeats,and insertion elements such as Alu. The first identified allelic form isarbitrarily designated as the “reference form” or “reference allele” andother allelic forms are designated as alternative forms or “variantalleles”. The allelic form occurring most frequently in a selectedpopulation is sometimes referred to as the wild type form. Diploidorganisms may be homozygous or heterozygous for allelic forms. Adiallelic or biallelic polymorphism has two forms. A triallelicpolymorphism has three forms.

As used herein, the term “genotype” is meant to encompass the particularallele present at a polymorphic locus of a DNA sample, a gene, and/orchromosome.

As used herein, the term “haplotype” is meant to encompass thecombination of genotypes across two or more polymorphic loci of a DNAsample, a gene, and/or chromosome, wherein the genotypes are closelylinked, may be inherited together as a unit, and may be in linkagedisequilibrium relative to other haplotypes and/or genotypes of otherDNA samples, genes, and/or chromosomes.

As used herein, the term “linkage disequilibrium” refers to a measure ofthe degree of association between two alleles in a population. Forexample, when alleles at two distinctive loci occur in a sample morefrequently than expected given the known allele frequencies andrecombination fraction between the two loci, the two alleles may bedescribed as being in “linkage disequilibrium”.

As used herein, the terms “genotype assay” and “genotype determination”,and the phrase “to genotype” or the verb usage of the term “genotype”are intended to be equivalent and refer to assays designed to identifythe allele or alleles at a particular polymorphic locus or loci in a DNAsample, a gene, and/or chromosome. Such assays may employ single baseextension reactions, DNA amplification reactions that amplify across oneor more polymorphic loci, or may be as simple as sequencing across oneor more polymorphic loci. A number of methods are known in the art forgenotyping, with many of these assays being described herein or referredto herein.

Work described herein pertains to the resequencing of the human resistingene in a large number of individuals to identify polymorphismsassociated with the incidence of HIV-1 protease inhibitor-dependentmetabolic abnormalities disorders upon the administration of a HIV-1protease inhibitor, which may predispose individuals to developing sucha disorder. For example, polymorphisms in the resistin gene describedherein are associated with the incidence of HIV-1 proteaseinhibitor-dependent metabolic abnormalities disorders and are useful forpredicting the likelihood that an individual will be susceptible to sucha disorder, or that such an individual may have an increasedsusceptibility to such a disorder, upon the administration of a HIV-1protease inhibitor.

By altering amino acid sequence, SNPs may alter the function of theencoded proteins. The discovery of the SNP facilitates biochemicalanalysis of the variants and the development of assays to characterizethe variants and to screen for pharmaceutical compounds that wouldinteract directly with one or another form of the protein. SNPs(including silent SNPs) may also alter the regulation of the gene at thetranscriptional or post-transcriptional level. SNPs (including silentSNPs) also enable the development of specific DNA, RNA, or protein-baseddiagnostics that detect the presence or absence of the polymorphism inparticular conditions.

The phrase “HIV-1 protease inhibitor” is meant to encompass compounds,including, but not limited to, atazanavir, lopinovir, ritonavir,indinavir, saquinavir, ramelteon, tipranavir, nepafenac, deferasirox,fosamprenavir, amprenavir, brecanvir, nelfinavir, as well as anycombination and/or boosted formulation of the same.

The phrase “PPAR-agonist” is meant to encompass compounds, includingsmall molecules, antibodies, RNAi reagents, siRNA reagents, antisensecompounds, or any compound in general capable of increasing the activityor expression of one or more peroxisome proliferator activator receptors(PPAR), including but not limited to, PPAR-alpha agonists, PPAR-betaagonists, PPAR-gamma agonists, and PPAR-delta agonists, includingmono-PPAR-alpha agonists, mono-PPAR-beta agonists, mono-PPAR-gammaagonists, mono-PPAR-delta agonists, dual PPAR-alpha and gamma agonists,and any combination of the same. In addition, such PPAR-agonists arenecessarily meant to encompass the following, non-limiting compounds:Muraglitazar, peliglitazar, Farglitazar, thiazolidinediones class ofPPAR-agonists, Troglitazone, Pioglitazone, Rosiglitazone, MCC555,KRP297, JTT-501, BM 17.0744, L764486, GW501516, NN622, bezafibrate,gemfibrozil, fibrate class of PPAR-agonists, DRF 2725, WY 14,643, SB213068, Tesaglitazar (AZ 242), Avandaryl, Naveglitazar, Ragaglitazar(NN622), PLX 204, PLX 134, PLX 203, CS 7017, DRF 10945, AVE 0847, AVE8134, 641597 (GSK), 590735 (GSK), MK 767, AA 10090, LY 674, LY 929, T131, DRF 4158, CLX 0921, NS 220, LY 293111, DRF 4832, GW 7282, 501516(GSK), LG 100754, GW 544, AR H049020, AK-109, E-3030 (Eisai), CS-7017(Sankyo), DRF-10945, KRP-101, ONO-5129, TY-51501, GSK-677954, LSN-862,LY-518674, GW-590735, KT6-207, K-111 (Roche), Bay-54-9801 (GSK), R-483(Roche), EMD-336340 (Merck KGaA), LR-90 (Merck KGaA), CLX-0940,CLX-0921, LG-100754, GW-409890, SB-219994, NIP-223, T-174 (TanabeSeiyaku), balaglitazone (DRF-2593), VDO-52, GW-1929, NC-2100,netoglitazone, ciglitazone, LGD 1268, LG 101506, LGD 1324, GW 9578,Englitazone, and/or Darglitazone.

The phrases “a pharmaceutically acceptable amount of an agent thatinhibits resistin activity” and “a pharmaceutically acceptable amount ofan agent that decreases the expression level of resistin in a patient”are meant to encompass any inhibitor of resistin, including, but notlimited to, small molecule inhibitors, antisense oligonucleotides, RNAimolecules, siRNAi molecules, ribozymes, zinc finger proteins,antibodies, antibody fragments, among other inhibitors directed torenin, otherwise known in the art.

A single nucleotide polymorphism occurs at a polymorphic locus occupiedby a single nucleotide, which is the site of variation between allelicsequences. The site is usually preceded by and followed by highlyconserved sequences of the allele (e.g., sequences that vary in lessthan 1/100 or 1/1000 members of the populations).

A single nucleotide polymorphism usually arises due to substitution ofone nucleotide for another at the polymorphic locus. A transition is thereplacement of one purine by another purine or one pyrimidine by anotherpyrimidine. A transversion is the replacement of a purine by apyrimidine or vice versa. Single nucleotide polymorphisms can also arisefrom a deletion of a nucleotide or an insertion of a nucleotide relativeto a reference allele. Typically the polymorphic locus is occupied by abase other than the reference base. For example, where the referenceallele contains the base “C” at the polymorphic site, the altered allelecan contain a “T”, “G” or “A” at the polymorphic locus.

For the purposes of the present invention the terms “polymorphicposition”, “polymorphic site”, “polymorphic locus”, and “polymorphicallele” shall be construed to be equivalent and are defined as thelocation of a sequence identified as having more than one nucleotiderepresented at that location in a population comprising at least one ormore individuals, and/or chromosomes.

Probe hybridizations are usually performed under stringent conditions,for example, at a salt concentration of no more than 1 M and atemperature of at least 25° C. For example, conditions of 5× SSPE (750mM NaCl, mM NaPhosphate, mM EDT A, pH 7.4) and a temperature of 25-30°C., or equivalent conditions, are suitable for allele-specific probehybridizations. Equivalent conditions can be determined by varying oneor more of the parameters given as an example, as known in the art,while maintaining a similar degree of identity or similarity between thetarget nucleotide sequence and the primer or probe used.

The term “isolated” is used herein to indicate that the material inquestion exists in a physical milieu distinct from that in which itoccurs in nature, and thus is altered “by the hand of man” from itsnatural state.

On one hand, and in specific embodiments, the polynucleotides of theinvention are at least 15, at least 30, at least 50, at least 100, atleast 125, at least 500, or at least 1000 continuous nucleotides but areless than or equal to 300 kb, 200 kb, 100 kb, 50 kb, 15 kb, 10 kb, 7.5kb, 5 kb, 2.5 kb, 2.0 kb, or 1 kb, in length. In a further embodiment,polynucleotides of the invention comprise a portion of the codingsequences, as disclosed herein, and may comprise all or a portion of anintron. In another embodiment, the polynucleotides preferentially do notcontain the genomic sequence of the gene or genes flanking the humanresistin (i.e., 5′ or 3′ to the resistin gene in the genome). In otherembodiments, the polynucleotides of the invention do not contain thecoding sequence of more than 1000, 500, 250, 100, 50, 25, 20, 15, 10, 5,4, 3, 2, or 1 genomic flanking gene(s).

On the other hand, and in specific embodiments, the polynucleotides ofthe invention are at least 15, at least 30, at least 50, at least 100,at least 125, at least 500, or at least 1000 continuous nucleotides butare less than or equal to 300 kb, 200 kb, 100 kb, 50 kb, 15 kb, 10 kb,7.5 kb, 5 kb, 2.5 kb, 2.0 kb, or 1 kb, in length. In a furtherembodiment, polynucleotides of the invention comprise a portion of thecoding sequences, comprise a portion of non-coding sequences, comprise aportion of an intron sequence, etc., or any combination of the latter,as disclosed herein. Alternatively, the polynucleotides of the inventionmay comprise the entire coding sequence, the entire 5′ non-codingsequence, the entire 3′ non-coding sequence, an entire intron sequence,an entire exon sequence, or any combination of the latter, as disclosedherein. In another embodiment, the polynucleotides may correspond to agenomic sequence flanking a gene (i.e., 5′ or 3′ to the gene of interestin the genome). In other embodiments, the polynucleotides of theinvention may contain the non-coding sequence of more than 1000, 500,250, 100, 50, 25, 20, 15, 10, 5, 4, 3, 2, or 1 genomic flanking gene(s).

As used herein, a “polynucleotide” refers to a molecule comprising anucleic acid of SEQ ID NO:1 or 2. For example, the polynucleotide cancontain the nucleotide sequence of the full length cDNA sequence,including the 5′ and 3′ untranslated sequences, the coding region, withor without a signal sequence, the secreted protein coding region, andthe genomic sequence with or without the accompanying promoter andtranscriptional termination sequences, as well as fragments, epitopes,domains, and variants of the nucleic acid sequence. Moreover, as usedherein, a “polypeptide” refers to a molecule having the translated aminoacid sequence generated from the polynucleotide as defined.

Unless otherwise indicated, all nucleotide sequences determined bysequencing a DNA molecule herein were determined using an automated DNAsequencer (such as the Model 3730-XL from Applied Biosystems, Inc.,and/or other PE 9700 from Perkin Elmer), and all amino acid sequences ofpolypeptides encoded by DNA molecules determined herein were predictedby translation of a DNA sequence determined above. The nucleotidesequence can also be determined by other approaches including manual DNAsequencing methods well known in the art. As is also known in the art, asingle insertion or deletion in a determined nucleotide sequencecompared to the actual sequence will cause a frame shift in translationof the nucleotide sequence such that the predicted amino acid sequenceencoded by a determined nucleotide sequence will be completely differentfrom the amino acid sequence actually encoded by the sequenced DNAmolecule, beginning at the point of such an insertion or deletion. Sincethe present relates to the identification of single nucleotidepolymorphisms whereby the novel sequence differs by as few as a singlenucleotide from a reference sequence, identified SNPs were multiplyverified to ensure each novel sequence represented a true SNP.

Using the information provided herein, a nucleic acid molecule of thepresent invention encoding a polypeptide of the present invention may beobtained using standard cloning and screening procedures, such as thosefor cloning cDNAs using mRNA as starting material.

A “polynucleotide” of the present invention also includes thosepolynucleotides capable of hybridizing, under stringent hybridizationconditions, to sequences described herein, or the complement thereof.“Stringent hybridization conditions” refers to an overnight incubationat 42° C. in a solution comprising 50% formamide, 5× SSC (750 mM NaCl,75 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5× Denhardt'ssolution, 10% dextran sulfate, and 20 μg/ml denatured, sheared salmonsperm DNA, followed by washing the filters in 0.1×SSC at about 65° C.

The polynucleotide of the present invention can be composed of anypolyribonucleotide or polydeoxribonucleotide, which may be unmodifiedRNA or DNA or modified RNA or DNA. For example, polynucleotides can becomposed of single- and double-stranded DNA, DNA that is a mixture ofsingle- and double-stranded regions, single- and double-stranded RNA,and RNA that is mixture of single- and double-stranded regions, hybridmolecules comprising DNA and RNA that may be single-stranded or, moretypically, double-stranded or a mixture of single- and double-strandedregions. In addition, the polynucleotide can be composed oftriple-stranded regions comprising RNA or DNA or both RNA and DNA. Apolynucleotide may also contain one or more modified bases or DNA or RNAbackbones modified for stability or for other reasons. “Modified” basesinclude, for example, tritylated bases and unusual bases such asinosine. A variety of modifications can be made to DNA and RNA; thus,“polynucleotide” embraces chemically, enzymatically, or metabolicallymodified forms.

The term “organism” as referred to herein is meant to encompass anyorganism referenced herein, though preferably to eukaryotic organisms,more preferably to mammals, and most preferably to humans.

As used herein the terms “modulate” or “modulates” refer to an increaseor decrease in the amount, quality or effect of a particular activity,DNA, RNA, or protein. The definition of “modulate” or “modulates” asused herein is meant to encompass agonists and/or antagonists of aparticular activity, DNA, RNA, or protein.

The phrase “HIV-1 protease inhibitor-dependent metabolic abnormalities”is meant to encompass the following non-limiting diseases and/ordisorders: HAART-associated lipodystrophy, lipodystrophy, fat atrophy,fat hypertrophy, dyslipidemia, insulin resistance, cardiovasculardisorders, myocardial infarction, stroke, atherosclerosis, peripherallipoatrophy, central fat accumulation, hepatic steatosis, lipodystrophysyndrome, lipodystrophy-lie syndromes, hyperglycemia, decreased HDLlevels, elevated levels of VLDL, hypertriglyceridemia,hypercholesterolemia, and hyperlipidemia. The definition necessarilyencompasses any other diseases and/or disorders that are known to beassociated with HIV-1 protease inhibitor administration, in addition toother diseases or disorders disclosed herein.

The term “HAART” is a reference to highly active antiretroviral therapyfor the treatment of HIV-1 infection. HAART therapy typicallyencompasses a double nucleoside (NRTI) backbone plus either anon-nucleoside reverse transcriptase inhibitor (NNRTI) or a ritonavirpharmacologically enhanced protease inhibitor (PI/r). However the actualtherapeutic composition in terms of both class and active agent variesdepending upon availability of each agent and a patients individualtolerance for each ingredient, among others. Accordingly, use of theterm “HAART” is meant to broadly encompass all combinations of activetherapeutic agents that the art would ascribe to this term.

The terms “1398T” and “C1398T” are meant to refer to the “t” allele atthe polymorphic locus located at nucleotide 1398 of SEQ ID NO:1. Oneskilled in the art would recognize that reference to this allele is notlimited to only SEQ ID NO:1, but rather necessarily also includes anyother polynucleotide that may include this sequence, or a portion ofthis sequence surrounding this locus, on account of SEQ ID NO:1 merelyrepresenting a small portion of chromosome 19 encoding the resistingene.

The term “C1398” is meant to refer to the “c” allele at the polymorphiclocus located at nucleotide 1398 of SEQ ID NO:1. One skilled in the artwould recognize that reference to this allele is not limited to only SEQID NO:1, but rather necessarily also includes any other polynucleotidethat may include this sequence, or a portion of this sequencesurrounding this locus, on account of SEQ ID NO:1 merely representing asmall portion of chromosome 19 encoding the resistin gene.

Methods of Screening for HIV-1 Protease Inhibitors with DiminishedAbility to Proteotyically Process Resistin

As part of an ongoing effort to understand the mechanistic basisunderlying HAART-associated lipodystrophy, resistin was evaluated as asubstrate for the endogenous aspartyl proteases renin, cathepsin D,cathepsin E for the viral aspartyl protease encoded by HIV-1.

Resistin was chosen as a candidate substrate based upon genetic datathat associated a single nucleotide polymorphism in resistin with therisk of developing lipodystrophy in patients enrolled on HAART therapy.The resistin SNP is described elsewhere herein.

There are at least two, non mutually exclusive, mechanisms that couldaccount for elevated resistin levels in patients on HAART. Because HIVinfection itself is considered to be a state of chronic inflammation, itis possible that HIV positive patients express higher circulating levelsof resistin compared to healthy subjects. Indeed, it has beendemonstrated in vitro and in vivo that treatment with pro-inflammatorycytokines such as interleukin 6, TNF, as well as lipopolysaccharideresults in increased resistin expression (Kaser, S., et al., BiochemBiophys Res Commun, 309(2):286-90 (2003), Lehrke, M., et al., PLoS Med,1(2):e45 (2004)). Furthermore, in patients treated with HAART, it isconceivable that inhibition of endogenous proteases that regulatesteady-state levels of resistin could result in increased circulatingresistin levels. In genetically susceptible patients, these mechanismscould contribute to lipodystrophy syndrome.

Resistin is known to form highly-folded, multimers that are maintainedvia extensive disulfide bridging (Patel, S. D., et al., Science,304(5674):1154-8 (2004)). Proteins exhibiting these properties aretypically refractory to proteolytic cleavage (Patel, S. D., et al.,Science, 304(5674): 1154-8 (2004)). Consistent with that expectation,preliminary protease digestion experiments revealed that native resistinwas refractory to proteolytic cleavage by trypsin, suggestive of ahighly folded state (data not shown). Therefore, to evaluate additionalproteases, resistin was fully denatured via alkylation and reduction ofcysteine residues.

Aspartyl proteases such as cathepsin D and cathepsin E are used tocounter screen HIV-1 protease inhibitors for selectivity toward HIV-1protease. It is well-established that these enzymes display relaxedsubstrate specificity (Dash, C., et al., Crit Rev Biochem Mol Biol,38(2):89-119 (2003), Chou, K. C., et al., Proteins, 24(1): p. 51-72(1996)), whereas others, such as renin, display more stringent substratespecificity (Dash, C., et al., Crit Rev Biochem Mol Biol, 38(2):89-119(2003)). Four aspartyl proteases, HIV-1 protease, cathepsin D, cathepsinE and renin were evaluated for their ability to cleave resistin (MW ca.10.4 kDa) in vitro. Incubation with HIV-1 protease, cathepsin D andcathepsin E resulted in the appearance of a ca. 7 kDa resistin cleavageproduct (see FIG. 1). Additional cleavage products were not detectableby SDS-PAGE, suggesting that the remaining ca. 3.4 kDa fragment wasfurther proteolyzed. Consistent with expectation, resistin wascompletely degraded by the non-specific protease, trypsin, but wasrefractory to cleavage by the highly specific protease, renin.

The ability of resistin to be proteolytically degraded by the aspartylproteases cathepsin D and E represents a novel finding and provides aunique insight into the physiological regulation of resistin levels. Theconnection between resistin, cathepsins D and E, and HAART therapybecomes apparent due to the discovery that some HIV-1 proteaseinhibitors vary substantially in their selectivity toward HIV-1 protease(i.e. they are also capable of inhibiting cathepsin D and cathepsin E,(Int J Biochem Cell Biol, 36(9):1787-99 (2004)).

The inventors therefore evaluated the protease inhibitors atazanavir andritonavir for their ability to inhibit the proteolysis of resistin bycathepsin D and cathepsin E. Cleavage of resistin by cathepsin D andcathepsin E was not detectably inhibited by atazanavir, however,cleavage of resistin by cathepsin D and cathepsin E was detectablyinhibited by ritonavir at 30 micromolar and 10 micromolar, respectively(see FIG. 2). Cathepsin D and cathepsin E were fully inhibited byritonavir at 100 micromolar and 30 micromolar, respectively. Consistentwith expectation, control experiments revealed that all three proteaseinhibitors antagonized HIV-1 protease-dependent cleavage of resistin atall concentrations evaluated. As expected, trypsin, an unrelated serineprotease, was not detectably inhibited by any protease inhibitor in thisstudy (see FIG. 2).

Additional HIV-1 protease inhibitors were also evaluated in parallel forcomparison, including nelfinavir, saquinavir and indinavir. In furthersupport of the hypothesis that inhibition of resistin cleavage bycathepsins D and E is predictive of HIV-1 protease inhibitors that aremore likely to cause metabolic abnormalities, cleavage of resistin bycathepsins D and E was prevented by saquinavir, but not by nelfinavir orindinavir (data not shown). Saquinavir is known to be associated withthe incidence of HIV-1 protease inhibitor induced metabolicabnormalities (Calza et al., J Antimicrob Chemother. 53(1):10-4 (2004),whereas nelfinavir (Fisac et al., J Clin Endocrinol Metab.88(11):5186-92 (2003)), or indinavir (Rojas et al., PharmacoepidemiolDrug Saf. 12(5):361-9 (2003)) are not.

The sensitivity of cathepsin D and cathepsin E to ritonavir, and theproposed effect of the latter on elevated resistin levels, is indirectlysupported by clinical trials demonstrating administration of ritonaviris associated with pronounced metabolic abnormalities relative to otherprotease inhibitors. Periard et al. show that elevated plasmacholesterol and plasma triglyceride levels were significantly morepronounced in patients administered ritonavir relative to indinavir ornelfinavir (Circulation, 100:700-705 (1999)). Manfredi, R. and Chiodo,F. demonstrated that administration of ritonavir was associated withsevere hypertriglyceridaemia compared to patients administeredindinavir, while hypercholesterolemia was observed in patientsadministered ritonavir and indinavir relative to saquinavir (J. Infect.,42:181-188 (2001)). Calza et al., also stated that“hypertriglyceridaemia appear(ed) to be more frequent in patientstreated with ritonavir, rinonavir-saquinavir, or ritonavir-lopinovir”(J. Antimicro. Chemo., 53:10-14 (2004).

In addition, atazanavir is known in the art to not result in significantmetabolic changes. Cohen et al., describe the results of a head-to-headclinical trial comparing atazanavir with lopinovir/ritonavir therapy andnoted that “atazanavir resulted in either no change or decreases infasting LDL cholesterol, total cholesterol, and fasting triglycerides .. . , whereas lopinovir/ritonavir resulted in increases . . . ” (Curr.Med. Res. Opin., 21(10):1683-92 (2005)). Further, Goldsmith et al.states that “Atazanavir was not associated with increases in totalcholesterol, low density lipoprotein-cholesterol or triglyceride levelsafter 108 weeks” in another clinical trial (Drugs, 63(16):1679-93(2003)).

Although clinical trials directed at assessing the metabolic affects oflopinovir alone appear to be limited because it is typicallyadministered in combination with ritonavir, at least two independentgroups published results that indicated lipid abnormalities did notcorrelate with lopinovir plasma concentrations suggesting the lipidaffects were attributable to ritonavir (J. Acquir. Immune. Defic. Syndr.35(3):324-6 (2004); and J. Acquir. Immune. Defic. Syndr. 36(5):1107-9(2004)).

Therefore, since HIV-1 protease inhibitors vary in their potential tocontribute toward the metabolic side-effects associated withlipodystrophy syndrome (Carr, A., Clin Infect Dis, 30 Suppl 2:S135-42(2000)), it would be of utility to be able to screen preclinicalcompounds to reduce the likelihood of a patient developing HIV-1protease inhibitor-dependent metabolic abnormalities. In this study, theinventors have demonstrated that the HIV-1 protease inhibitorsatazanavir, ritonavir and lopinovir differentially inhibit cleavage ofresistin by cathepsin D and cathepsin E. Because resistin is associatedwith obesity and insulin resistance (Rajala, M. W., et al., J ClinInvest, 11 1(2):225-30 (2003)), and because HIV-1 protease inhibitortherapy can cause hyperlipidemia (Carr, A., Clin Infect Dis, 30 Suppl2:S135-42 (2000)), it is conceivable, that inhibition of cathepsins Dand E by HIV-1 protease inhibitors, may result in elevated resistinlevels. In genetically susceptible HIV-1 positive patients on HAARTtherapy, this may ultimately lead to components of lipodystrophysyndrome and/or metabolic abnormalities, in general.

The present invention relates to a method for screening for HIV-1protease inhibitor compounds with a diminished ability to increase anindividuals likelihood of developing HIV-1 protease inhibitor-dependentmetabolic abnormalities upon the administration of the same. In specificembodiments of the invention, such a method comprises the step ofcombining a mixture of resistin and cathepsin D or E with either one ormore test drugs, compounds, or other therapeutic agents, or a controlcompound; followed by the step of measuring the level of proteolyzedresistin between the test and control samples, and selecting the testcompound with a diminished ability to inhibit cathepsin D- or cathepsinE-dependent degradation of resistin relative to a control compound.

The present invention relates to methods of predicting whether a patienthas an increased risk of developing HIV-1 protease inhibitor-dependentmetabolic abnormalities upon the administration of a pharmaceuticallyacceptable level of a HIV-1 protease inhibitor; whether said patientrequires a lower level of administered HIV-1 protease inhibitor to limitthe risk of developing said HIV-1 protease inhibitor-dependent metabolicabnormalities; or whether said patient may be administered a higherlevel of administered HIV-1 protease inhibitor without the risk or of alower risk of developing said HIV-1 protease inhibitor-dependentmetabolic abnormalities, comprising the step of assessing the level ofresistin expression or resistin plasma/serum levels resulting from theadministration of a HIV-1 protease inhibitor relative to a controlcompound, wherein an elevated level of resistin expression or resistinplasma/serum levels is indicative of an increased risk of developingHIV-1 protease inhibitor-dependent metabolic abnormalities subsequent tothe administration of a HIV-1 protease inhibitor.

In addition, the expression level or plasma/serum level of humanresistin is believed to be significantly elevated in the presence ofHIV-1 protease inhibitors that are capable of inhibiting cathepsin Dand/or cathepsin E thus suggesting that human resistin may be useful asa biomarker for predicting whether a patient administered an HIV-lprotease inhibitor may develop metabolic abnormalities. In accordancewith the present invention, an elevated basal level of resistin prior tothe administration of an HIV-1 protease inhibitor would be indicative ofan increased risk of developing metabolic abnormalities subsequent tothe administration of an HIV-1 protease inhibitor. In addition, anelevated basal level of resistin prior to the administration of an HIV-1protease inhibitor would be indicative of an even greater increased riskof developing metabolic abnormalities subsequent to the administrationof an HIV-1 protease inhibitor capable of inhibiting cathepsin D and/orcathepsin E.

An elevated basal resistin plasma/serum level in a patient may warrantcloser monitoring of the patient prior to increasing the level ofadministered HIV-1 protease inhibitor to limit the risk of developingsaid HIV-1 protease inhibitor-dependent metabolic abnormalities,particularly if an HIV-1 protease inhibitor capable of inhibitingcathepsin D and/or cathepsin E is administered. Accordingly, an elevatedbasal resistin plasma/serum level in a patient may warrant lowering theamount of the HIV-1 protease inhibitor administered or changing toanother HIV-1 protease inhibitor that does not inhibit cathespin Dand/or cathepsin E, or at least inhibits cathespin D and/or cathepsin Eto a lesser extent. Alternatively, basal or normal plasma/serum levelsof resistin in a patient may justify the administration of a higherlevel of HIV-1 protease inhibitor with a diminished risk of developingHIV-1 protease inhibitor-dependent metabolic abnormalities.Alternatively, elevated basal resistin plasma/serum level in a patientmay indicate that the patient may benefit from hypertriglyceridemiatherapy which may include administration of bezafibrate either alone orin combination with an HIV-1 protease inhibitor.

In another embodiment of the present invention, human resistin is usefulas a biomarker for pre- or post-clinical screening to identify HIV-1protease inhibitor compounds or combinations of such compounds that arelikely to increase the risk of a patient developing HIV-1 proteaseinhibitor-dependent metabolic abnormalities in response to theadministration of HIV-1 protease inhibitor compounds or combinations ofsuch compounds, and thus to prevent or diminish the likelihood of apatient developing HIV-1 protease inhibitor-dependent metabolicabnormalities by either advising patients be monitored more closely ifsuch a compound or combination of compounds are administered at acorresponding higher dose, or by changing the amount of HIV-1 proteaseinhibitor administered or the composition of the combination.Preferably, such an identified HIV-1 protease inhibitor compound orcombinations of such compounds would not be capable of inhibitingcathepsin D and/or cathepsin E.

Cells endogenously expressing human resistin can be treated with atleast one test substance, and extracellular and/or intracellular levelsof the biomarker resistin polypeptide in the presence and absence of thetest substance(s) can be compared. The observation of high levels of theresistin biomarker polypeptide in the presence of the substance(s) canbe used to predict which compounds are likely to increase the risk of apatient developing HIV-1 protease inhibitor-dependent metabolicabnormalities in response to the administration of HIV-1 proteaseinhibitor compounds or combinations of such compounds, and thus toprevent or diminish the likelihood of a patient developing HIV-1protease inhibitor-dependent metabolic abnormalities by not selectingsuch a test compound in the screen. In an additional aspect, the assaysof the invention are automated for high throughput screening. Theresults of such screening may be used to determine the need to modify ordiscontinue an existing treatment.

The present invention also encompasses microarrays, e.g., protein,antibody, or cell-based microarrays, which can be used in conjunctionwith the disclosed screening assays for measuring the resistin biomarkerpolypeptide. The protein, antibody, and cell-based microarrays can beused in the manual or automated screening assays of the invention asdisclosed herein to test one or more drugs, compounds, or othertherapeutic agents. For protein microarrays, polypeptides obtained fromresistin expressing cells (e.g., from extracellular media or celllysates) incubated in the presence and absence of at least one testsubstance can be affixed to a support, and then contacted withantibodies that specifically bind to the resistin biomarker polypeptide.For antibody microarrays, one or more anti-biomarker antibodies can beaffixed to a support, and then contacted with extracellular media orcell lysates obtained from resistin expressing cells incubated in thepresence and absence of at least one test substance. For cell-basedmicroarrays, one or more cells can be affixed to a support, and thenincubated in the presence and absence of at least one test substance.The microarrays can then be analyzed (e.g., by immunoassay) to determineelevated levels of at least one biomarker polypeptide in the presence ofthe test substance(s), which can be used to predict which compound arelikely to increase the risk of a patient developing HIV-1 proteaseinhibitor-dependent metabolic abnormalities in response to theadministration of HIV-1 protease inhibitor compounds or combinations ofsuch compounds, and thus to prevent or diminish the likelihood of apatient developing HIV-1 protease inhibitor-dependent metabolicabnormalities by either decreasing the level of the administered HIV-1protease inhibitor compounds or combinations of such compounds, or bychanging the HIV-1 protease inhibitor combination administered.

The present invention additionally encompasses kits comprising one ormore biomarker polypeptides, and/or anti-biomarker antibodies, which canbe used to predict the likelihood of HIV-1 protease inhibitor-dependentmetabolic abnormalities of one or more drugs, compounds, or othertherapeutic agents. Such kits can be used in clinical or pre-clinicalsettings, and can include one or more biomarker polypeptides andanti-biomarker antibodies. In specific aspects of the invention, thekits can include one or more microarrays comprising antibodies thatspecifically bind with these biomarker polypeptides. The kits can beemployed in conjunction with the manual and automated screening methodsof the invention. In various aspects, the kits can include instructionsfor use, and reagents and materials for measuring levels of thebiomarker polypeptides e.g., in immunoassays, such as enzyme linkedimmunosorbent assays (ELISAs); Western blotting; direct or indirectimmunofluorescence, immunohistochemistry, and the like.

The present invention further encompasses cell culture systems for theidentification of polypeptides, in addition to the specified biomarkerpolypeptides, whose levels (e.g., extracellular, intracellular, or celllysate levels) correlate with increased risk of developing HIV-1protease inhibitor-dependent metabolic abnormalities upon theadministration of a HIV-1 protease inhibitor. In specific aspects of theinvention, such systems can comprise resistin expressing cell lines,which can be incubated in the presence or absence of one or more drugs,compounds, or other therapeutic agents. The biomarker polypeptidesidentified from these systems can be useful for identifying testsubstances (or combinations of test substances) that may directly orindirectly increase the risk of a patient developing HIV-1 proteaseinhibitor-dependent metabolic abnormalities in response to theadministration of HIV-1 protease inhibitor compounds or combinations ofsuch compounds, and thus to prevent or diminish the likelihood of apatient developing HIV-1 protease inhibitor-dependent metabolicabnormalities by either decreasing the level of the administered HIV-1protease inhibitor compounds or combinations of such compounds, or bychanging the HIV-1 protease inhibitor combination administered.

The present invention encompasses methods of measuring the levels ofpolypeptides (e.g., extracellular polypeptides in the media) using massspectrometry data to determine the number of peptide “hits” for eachpolypeptide, and comparing the results obtained in the presence andabsence of a test substance.

Also encompassed by the invention are nucleic acids encoding thedisclosed resistin biomarker polypeptide (SEQ ID NO:12), and fragments,variants, and derivatives thereof, as well as screening assays, kits,microarrays, and cell culture systems employing these nucleic acids. Inone aspect of the invention, screening assays (e.g., RT-PCR or in situassays) that measure levels of one or more biomarker nucleic acids areused to predict which compound are likely to increase the risk of apatient developing HIV-1 protease inhibitor-dependent metabolicabnormalities in response to the administration of HIV-1 proteaseinhibitor compounds or combinations of such compounds, and thus toprevent or diminish the likelihood of a patient developing HIV-1protease inhibitor-dependent metabolic abnormalities by eitherdecreasing the level of the administered HIV-1 protease inhibitorcompounds or combinations of such compounds, or by changing the HIV-1protease inhibitor combination administered. Elevated levels of one ormore biomarker nucleic acids in the presence of the test substance(s)can be used to predict which patients have an increased risk ofdeveloping HIV-1 protease inhibitor-dependent metabolic abnormalities inresponse to the administration of HIV-1 protease inhibitor compounds orcombinations of such compounds, and thus to identify those patients thatrequire monitoring more closely if an increased dosage of a HIV-1protease inhibitor is contemplated in order to avoid the potential ofincreasing the likelihood of developing HIV-1 proteaseinhibitor-dependent metabolic abnormalities. Alternatively, low levelsof one or more biomarker nucleic acids in the presence of the testsubstance(s) can be used to predict which patients have a decreased riskof developing HIV-1 protease inhibitor-dependent metabolic abnormalitiesin response to the administration of HIV-1 protease inhibitor compoundsor combinations of such compounds, and thus identify which patients maybe administered a correspondingly higher amount of a HIV-1 proteaseinhibitor without increasing the likelihood of developing HIV-1 proteaseinhibitor-dependent metabolic abnormalities.

The present invention also encompasses a method of predicting the riskthat compound may increase the risk of a patient developing HIV-1protease inhibitor-dependent metabolic abnormalities of a test substancecomprising the steps of: a) incubating a resistin expressing cell in thepresence and absence of a test substance; and b) comparing levels of atleast one biomarker polypeptide, in the presence and absence of saidtest substance; wherein an elevated level of said biomarkerpolypeptide(s) in the presence of the test substance indicates that thesubstance is predicted to increase the risk of a patient developingHIV-1 protease inhibitor-dependent metabolic abnormalities in responseto the administration of HIV-1 protease inhibitor compounds orcombinations of such compounds, and wherein the level of said biomarkerpolypeptide(s) is measured using single or multi dimensional highperformance liquid chromatography coupled to tandem mass spectrometrywherein the number of peptide hits from each protein identification areused to determine the abundance of said biomarker polypeptide(s) in thepresence and absence of said test substance.

Publications and other materials setting forth such the proteomicsmethodologies include the following: McDonald W H, Yates J R 3rd., 2002,Shotgun proteomics and biomarker discovery, Dis. Markers. 18(2):99-105;Link A J, 2002, Multidimensional peptide separations in proteomics,Trends Biotechnol. December;20(12 Suppl):S8-13. Additional publicationsoutlining the application of such proteomic methods is set forth in thefollowing: J. Gao et al, “Identification of In Vitro Protein Biomarkersof Idiosyncratic Liver Toxicity,” Toxicology In Vitro, 18(4), 533-541(2004); J. Gao et al, “Changes in the Protein Expression of Yeast as aFunction of Carbon Source,” Journal of Proteome Research, 2(6), 643-649(2003); J. X. Pang et al, “Biomarker Discovery in Urine by Proteomics,”Journal of Proteome Research, 1(2), 161-169 (2002). All of thesepublications are incorporated by reference herein in their entirety.

Polynucleotides and Polypeptides of the Invention Features of Gene No:1

The present invention relates to isolated nucleic acid moleculescomprising, or alternatively consisting of, all or a portion of one ormore alleles of SNP1 of the human resistin gene, as provided in FIGS.3A-B (SEQ ID NO:1) comprising at least one polymorphic locus. The alleledescribed for SNP1 in FIGS. 3A-B (SEQ ID NO:1) represents the referenceallele for this SNP and is exemplified by a “C” at nucleotide position1398. Fragments of this polynucleotide are at least about 10, at leastabout 20, at least about 40, or at least about 100, contiguousnucleotides and comprise one or more reference alleles at the nucleotideposition(s) provided in FIGS. 3A-B (SEQ ID NO:1).

In one embodiment, the invention relates to a method for predicting thelikelihood that an individual will have a disorder, particularly HIV-1protease inhibitor-dependent metabolic abnormalities, or be susceptibleto developing HIV-1 protease inhibitor-dependent metabolic abnormalitiesupon the administration of a pharmaceutically acceptable amount of anHIV-1 protease inhibitor, comprising the step of identifying thenucleotide present at nucleotide position 1398 of SEQ ID NO:1, from aDNA sample to be assessed, or the corresponding nucleotide at thisposition if only a fragment of the sequence provided as SEQ ID NO:1 isassessed. The presence of the reference allele at said positionindicates that the individual from whom said DNA sample or fragment wasobtained has a decreased likelihood of developing HIV-1 proteaseinhibitor-dependent metabolic abnormalities upon the administration of apharmaceutically acceptable amount of a HIV-1 protease inhibitor than anindividual having the variable allele(s) at said position(s); or andecreased likelihood of developing more severe HIV-1 proteaseinhibitor-dependent metabolic abnormalities upon administration of thesame.

Importantly, the presence of the reference allele at said position in anucleic acid sample provided by an individual, indicates that saidindividual may be administered a correspondingly higher amount of aHIV-1 protease inhibitor without increasing the likelihood of developingHIV-1 protease inhibitor-dependent metabolic abnormalities relative toanother individual having the variant allele(s) at said position.Therefore, such individuals may have the level of administered HIV-1protease inhibitor “titrated-up” or maintained at the normal levelwithout increasing the risk, or at least having a decreased risk, of apatient developing HIV-1 protease inhibitor-dependent metabolicabnormalities.

Representative disorders which may be detected, diagnosed, identified,treated, prevented, and/or ameliorated by the SNPs and methods of thepresent invention include, the following, non-limiting diseases anddisorders: HIV-1 protease inhibitor-dependent metabolic abnormalities,susceptibility to developing HIV-1 protease inhibitor-dependentmetabolic abnormalities upon the administration of a pharmaceuticallyacceptable amount of an HIV-1 protease inhibitor, susceptibility todeveloping HIV-1 protease inhibitor-dependent metabolic abnormalitiesupon the administration of an increased level of a HIV-1 proteaseinhibitor, adverse reactions associated with HIV-1 protease inhibitor,disorders associated with aberrant resistin expression, disordersassociated with aberrant resistin regulation, disorders associated withaberrant resistin activity, disorders associated with aberrantregulation of resistin by cathepsin, disorders associated with aberrantregulation of resistin by cathepsin D, disorders associated withaberrant regulation of resistin by cathepsin E, disorders associatedwith elevated resistin plasma/serum levels, HAART-associatedlipodystrophy, fat atrophy, fat hypertrophy, dyslipidemia, insulinresistance, cardiovascular disorders, myocardial infarction, stroke,atherosclerosis, peripheral lipoatrophy, central fat accumulation,hepatic steatosis, lipodystrophy syndrome, lipodystrophy-lie syndromes,hyperglycemia, decreased HDL levels, elevated levels of VLDL,hypertriglyceridemia, hypercholesterolemia, and hyperlipidemia.

Features of Gene No:2

The present invention relates to isolated nucleic acid moleculescomprising, or alternatively consisting of, all or a portion of one ormore alleles of SNP2 of the human resistin gene, as provided in FIGS.4A-B (SEQ ID NO:2) comprising at least one polymorphic locus. The alleledescribed for SNP2 in FIGS. 4A-B (SEQ ID NO:2) represents the variableallele for this SNP and is exemplified by an “T” at nucleotide position1398. Fragments of this polynucleotide are at least about 10, at leastabout 20, at least about 40, at least about 100, contiguous nucleotidesand comprise one or more variable alleles at the nucleotide position(s)provided in FIGS. 4A-B (SEQ ID NO:2).

In one embodiment, the invention relates to a method for predicting thelikelihood that an individual will have a disorder, particularly HIV-1protease inhibitor-dependent metabolic abnormalities, or be susceptibleto developing HIV-1 protease inhibitor-dependent metabolic abnormalitiesupon the administration of a pharmaceutically acceptable amount of aHIV-1 protease inhibitor, comprising the step of identifying thenucleotide present at nucleotide position 1398 of SEQ ID NO:2, from aDNA sample to be assessed, or the corresponding nucleotide at thisposition if only a fragment of the sequence provided as SEQ ID NO:2 isassessed. The presence of the variable allele at said position indicatesthat the individual from whom said DNA sample or fragment was obtainedhas an increased likelihood of developing HIV-1 proteaseinhibitor-dependent metabolic abnormalities upon the administration of apharmaceutically acceptable amount of a HIV-1 protease inhibitor,compared to an individual having the reference allele(s) at saidposition(s); or at least an increased likelihood of developing moresevere HIV-1 protease inhibitor-dependent metabolic abnormalities uponadministration of the same.

Importantly, the presence of the variable allele at said position in aDNA sample provided by an individual indicates that said individualshould be monitored more closely if an increased dosage of a HIV-1protease inhibitor is contemplated in order to avoid the potential ofincreasing the likelihood of developing HIV-1 proteaseinhibitor-dependent metabolic abnormalities relative to anotherindividual having the variant allele(s) at said position. In addition,the presence of the variable allele at said position in a DNA sampleindicates a lower dose of HIV-1 protease inhibitor may be warranted orthat it may be warranted to administer to the patient a different HIV-1protease inhibitor that does not have as much of a likelihood of causingHIV-1 protease inhibitor-dependent, metabolic abnormalities.Specifically, a patient harboring the variable allele, eitherheterozygously or homozygously, should be administered atazanavir, aprotease inhibitor less likely to cause metabolic abnormalities (Jemsek,J. G. et al., Clin Infect Dis. 42:273-80 (2006)), or administered one ormore PPARγ agonists, such as for example, rosiglitazone and/orpioglitazone, which are known to inhibit expression of resistin(Steppan, C. M. et al., Nature 409: 307-12 (2001), alone or incombination with HAART.

Alternatively, the presence of the variable allele at said position in aDNA sample provided by an individual indicates that the patient maybenefit from hypertriglyceridemia therapy which may includeadministration of bezafibrate either alone or in combination with anHIV-1 protease inhibitor (Ericsson, C G, J. Eur Heart, 19(SupplementA):A36-A39 (1998).

Representative disorders which may be detected, diagnosed, identified,treated, prevented, and/or ameliorated by the SNPs and methods of thepresent invention include, the following, non-limiting diseases anddisorders: HIV-1 protease inhibitor-dependent metabolic abnormalities,susceptibility to developing HIV-1 protease inhibitor-dependentmetabolic abnormalities upon the administration of a pharmaceuticallyacceptable amount of an HIV-1 protease inhibitor, susceptibility todeveloping HIV-1 protease inhibitor-dependent metabolic abnormalitiesupon the administration of an increased level of a HIV-1 proteaseinhibitor, adverse reactions associated with HIV-1 protease inhibitor,disorders associated with aberrant resistin expression, disordersassociated with aberrant resistin regulation, disorders associated withaberrant resistin activity, disorders associated with aberrantregulation of resistin by cathepsin, disorders associated with aberrantregulation of resistin by cathepsin D, disorders associated withaberrant regulation of resistin by cathepsin E, disorders associatedwith elevated resistin plasma/serum levels, HAART-associatedlipodystrophy, fat atrophy, fat hypertrophy, dyslipidemia, insulinresistance, cardiovascular disorders, myocardial infarction, stroke,atherosclerosis, peripheral lipoatrophy, central fat accumulation,hepatic steatosis, lipodystrophy syndrome, lipodystrophy-lie syndromes,hyperglycemia, decreased HDL levels, elevated levels of VLDL,hypertriglyceridemia, hypercholesterolemia, and hyperlipidemia.

TABLE I Polynucleotide CDNA Polymorphic Nucleotide Position ofNucleotide at SEQ ID No. CloneID Allele Locus Number Polymorphic LocusPolymorphic Locus NO: 1 Human Resistin Reference 1 1398 C 1 Gene - SNP12 Human Resistin Variable 1 1398 T 2 Gene - SNP1

The present invention provides a polynucleotide comprising, oralternatively consisting of, the sequence identified as SEQ ID NO:1and/or 2; or a fragment containing the polymorphic allele, wherein saidfragment comprises at least 10 contiguous nucleotides of SEQ ID NO:1and/or 2.

Preferably, the present invention is directed to a polynucleotidecomprising, or alternatively consisting of, the sequence identified asSEQ ID NO:1 and/or 2, that is less than, or equal to, a polynucleotidesequence that is 5 mega basepairs, 1 mega basepairs, 0.5 mega basepairs,0.1 mega basepairs, 50,000 basepairs, 20,000 basepairs, or 10,000basepairs in length.

The present invention encompasses polynucleotides with sequencescomplementary to those of the polynucleotides of the present inventiondisclosed herein. Such sequences may be complementary to the sequencedisclosed as SEQ ID NO:1 and/or 2.

The invention encompasses the application of PCR methodology to thepolynucleotide sequences of the present invention, and/or the cDNAencoding the polypeptides of the present invention. PCR techniques forthe amplification of nucleic acids are described in U.S. Pat. No.4,683,195 and Saiki et al., Science, 239:487-491 (1988). PCR, forexample, may include the following steps, of denaturation of templatenucleic acid (if double-stranded), annealing of primer to target, andpolymerization. The nucleic acid probed or used as a template in theamplification reaction may be genomic DNA, cDNA, RNA, or a PNA. PCR maybe used to amplify specific sequences from genomic DNA, specific RNAsequence, and/or cDNA transcribed from mRNA. References for the generaluse of PCR techniques, including specific method parameters, includeMullis et al., Cold Spring Harbor Symp. Quant. Biol., 51:263, (1987),Ehrlich (ed), PCR Technology, Stockton Press, NY, 1989; Ehrlich et al.,Science, 252:1643-1650, (1991); and “PCR Protocols, A Guide to Methodsand Applications”, Eds., Innis et al., Academic Press, New York, (1990).

Polynucleotide Variants

The present invention also encompasses variants (e.g., allelic variants,orthologs, etc.) of the polynucleotide sequence disclosed herein in SEQID NO:1 and/or 2, and the complementary strand thereto.

The present invention also encompasses variants of the polypeptidesequence, and/or fragments therein, disclosed in SEQ ID NO:12, apolypeptide encoded by the polynucleotide sequence in SEQ ID NO:1 and/or2.

“Variant” refers to a polynucleotide or polypeptide differing from thepolynucleotide or polypeptide of the present invention, but retainingessential properties thereof. Generally, variants are overall closelysimilar, and, in many regions, identical to the polynucleotide orpolypeptide of the present invention.

In another embodiment, the invention encompasses nucleic acid moleculeswhich comprise, or alternatively, consist of a polynucleotide whichhybridizes under stringent conditions, or alternatively, under lowerstringency conditions, to a polynucleotide in (a), (b), (c), or (d),above. Polynucleotides which hybridize to the complement of thesenucleic acid molecules under stringent hybridization conditions oralternatively, under lower stringency conditions, are also encompassedby the invention, as are polypeptides encoded by these polypeptides.

Polynucleotide Fragments

The present invention is directed to polynucleotide fragments of thepolynucleotides of the invention, and polynucleotide sequences thathybridize thereto.

In the present invention, a “polynucleotide fragment” refers to a shortpolynucleotide having a nucleic acid sequence which: is a portion ofthat shown in SEQ ID NO:1 and/or 2 or the complementary strand thereto,or is a portion of a polynucleotide sequence encoding the polypeptide ofSEQ ID NO:12. The nucleotide fragments of the invention are preferablyat least about 15 nt, and more preferably at least about 20 nt, stillmore preferably at least about 30 nt, and even more preferably, at leastabout 40 nt, at least about 50 nt, at least about 75 nt, or at leastabout 150 nt in length, and comprise at least one polymorphic locus. Afragment “at least 20 nt in length,” for example, is intended to include20 or more contiguous bases from the cDNA sequence shown in SEQ ID NO:1and/or 2. In this context “about” includes the particularly recitedvalue, a value larger or smaller by several (5, 4, 3, 2, or 1)nucleotides, at either terminus, or at both termini. These nucleotidefragments have uses that include, but are not limited to, as diagnosticprobes and primers as discussed herein. Of course, larger fragments(e.g., 50, 150, 500, 600, 2000 nucleotides) are preferred.

Moreover, representative examples of polynucleotide fragments of theinvention, include, for example, isolated fragments comprising, oralternatively consisting of, a sequence from about nucleotide number1-50, 51-100, 101-150, 151-200, 201-250, 251-300, 301-350, 351-400,401-450, 451-500, 501-550, 551-600, 651-700, 701-750, 751-800, 800-850,851-900, 901-950, 951-1000, 1001-1050, 1051-1100, 1101-1150, 1151-1200,1201-1250, 1251-1300, 1301-1350, 1351-1400, 1401-1450, 1451-1500,1501-1550, 1551-1600, 1601-1650, 1651-1700, 1701-1750, 1751-1800,1801-1850, 1851-1900, 1901-1950, 1951-2000, or 2001 to the end of SEQ IDNO:1 and/or 2, or the complementary strand thereto. In this context“about” includes the particularly recited ranges, and ranges larger orsmaller by several (5, 4, 3, 2, or 1) nucleotides, at either terminus orat both termini. Preferably, these fragments encode a polypeptide whichhas biological activity. More preferably, these polynucleotides can beused as probes or primers as discussed herein. Also encompassed by thepresent invention are polynucleotides which hybridize to these nucleicacid molecules under stringent hybridization conditions or lowerstringency conditions, as are the polypeptides encoded by thesepolynucleotides.

Kits

The invention further provides kits comprising at least one agent foridentifying which alleleic form of the SNPs identified herein is presentin a sample. For example, suitable kits can comprise at least oneantibody specific for a particular protein or peptide encoded by onealleleic form of the gene, or allele-specific oligonucleotide asdescribed herein. Often, the kits contain one or more pairs ofallele-specific oligonucleotides hybridizing to different forms of apolymorphism. In some kits, the allele-specific oligonucleotides areprovided immobilized to a substrate. For example, the same substrate cancomprise allele-specific oligonucleotide probes for detecting at least1, 10, 100 or all of the polymorphisms shown in Table I. Optionaladditional components of the kit include, for example, reagents,buffers, restriction enzymes, reverse-transcriptase or polymerase, thesubstrate nucleoside triphosphates, means used to label (for example, anavidin-enzyme conjugate and enzyme substrate and chromogen if the labelis biotin, fluophores, and others as described herein), and theappropriate buffers for reverse transcription, PCR, or hybridizationreactions. Usually, the kit also contains instructions for carrying outthe methods.

The present invention provides kits that can be used in the methodsdescribed herein. In one embodiment, a kit comprises a single primer orprobe of the invention comprising a means to detect at least onepolymorphic locus, said means preferably comprises a purified primer orprobe, in one or more containers. Such a primer or probe may furthercomprise a detectable label such as a fluorescent compound, an enzymaticsubstrate, a radioactive compound, a luminescent compound, afluorophore, and/or a fluorophore linked to a terminator containedtherein. Such a kit may further comprise reagents required to enableadequate hybridization of said single primer or probe to a DNA testsample, such that under suitable conditions, the primer or probe iscapable of binding to said DNA test sample and signaling whether thevariant or reference allele at the polymorphic locus is present in saidDNA test sample.

In one example, the kit comprises a means method for detecting thepresence of a polymorphic locus comprising one specific allele of atleast one polynucleotide in a DNA test sample which serves as a templatenucleic acid comprising: (a) forming an oligonucleotide bound to thepolymorphic locus wherein the oligonucleotide comprises a fluorophorelinked to a terminator contained therein; and (b) detecting fluorescencepolarization of the fluorophore of the fluorescently-labeledoligonucleotide, wherein the oligonucleotide is formed from a primerbound to said DNA sample immediately 3′ to the polymorphic locus and aterminator covalently linked to a fluorophore, and wherein saidterminator-linked fluorophore binds to the polymorphic locus and reactswith the primer to produce an extended primer which is saidfluorescently labeled oligonucleotide, wherein an increase influorescence polarization indicates the presence of the specific alleleat the polymorphic locus, thereby detecting the presence of the specificallele at the polymorphic locus by said increase in fluorescencepolarization.

The kit of the present invention may comprise the following non-limitingexamples of flurophores linked to a primer or probe of the presentinvention: 5-carboxyfluorescein (FAM-ddNTPs); 6-carboxy-X-rhodamine(ROX-ddNTPs); N,N,N′,N′-tetramethyl-6-carboxyrhodamine (TMR-ddNTPs); andBODIPY-Texas Red (BTR-ddNTPs).

The present invention is also directed towards a kit comprising a solidsupport to which oligonucleotides comprising at least 10 contiguousnucleotides of SEQ ID NO:1, 2, 3, 4, 5, 6, or 7, wherein saidoligonucleotide further comprises at least one polymorphic locus of SEQID NO:1, 2, 3, 4, 5, 6, or 7, are affixed. In such an embodiment,detection of a polynucleotide within a sample comprising the same orsimilar sequence to said oligonucleotide can be detected byhybridization.

The solid surface reagent in the above assay is prepared by knowntechniques for attaching protein material to solid support material,such as polymeric beads, dip sticks, 96-well plate or filter material.These attachment methods generally include non-specific adsorption ofthe oligonucleotide to the support or covalent attachment of theoligonucleotide to a chemically reactive group on the solid support.Alternatively, streptavidin coated plates can be used in conjunctionwith biotinylated oligonucleotide(s).

Thus, the invention provides an assay system or kit for carrying outthis diagnostic method. The kit generally includes. a support withsurface-bound oligonucleotides, and a reporter for detectinghybridization of said oligonucleotide to a test polynucleotide.

Methods of Use of the Allelic Polynucleotides of the Present Invention

The determination of the polymorphic form(s) present in an individual atone or more polymorphic sites defined herein can be used in a number ofmethods.

In preferred embodiments, the polynucleotides and polypeptides of thepresent invention, including allelic and variant forms thereof, haveuses which include, but are not limited to diagnosing individuals toidentify whether a given individual has increased susceptibility or riskfor developing HIV-1 protease inhibitor-dependent metabolicabnormalities disorder using the genotype assays of the presentinvention. In addition, the polynucleotides and polypeptides of thepresent invention, including allelic and variant forms thereof, haveuses which include, but are not limited to, diagnosing individuals toidentify whether a given individual, upon administration of an increaseddose of a HIV-1 protease inhibitor, has increased susceptibility or riskfor developing HIV-1 protease inhibitor-dependent metabolicabnormalities disorder using the genotype assays of the presentinvention.

Wherever the term “atazanavir” is used herein, it is understood (unlessotherwise indicated) that the compound “atazanavir” is intended as wellas all pharmaceutically acceptable salts thereof. Use of the termencompasses (unless otherwise indicated) solvates (including hydrates),crystal structures (including polymorphic forms of such structures) andsalts of the compound. Pharmaceutical compositions of atazanavir includeall pharmaceutically acceptable compositions comprising atazanavir andone or more diluents, vehicles and/or excipients.

Atazanavir is commercially available as a prescription medicine fromBristol-Myers Squibb Company, New York, under the tradename REYATAZ®(atazanavir sulfate) for the treatment of HIV. Approved in 2003 by theU.S. Food and Drug Administration, REYATAZ® (atazanavir sulfate) iscurrently available in the form of 100 milligram (“mg”), 150 mg, 200 mg,and 300 mg capsules.

U.S. Pat. No. 5,849,911 to Fassler et al. discloses a series ofazapeptide HIV protease inhibitors (which includes atazanavir) whichhave the structure

wherein

R₁ is lower alkoxycarbonyl,

R₂ is secondary or tertiary lower alkyl or lower alkylthio-lower alkyl,

R₃ is phenyl that is unsubstituted or substituted by one or more loweralkoxy radicals, or C₄-C₈ cycloalkyl,

R₄ is phenyl or cyclohexyl each substituted in the 4-position byunsaturated heterocyclyl that is bonded by way of a ring carbon atom,has from 5 to 8 ring atoms, contains from 1 to 4 hetero atoms selectedfrom nitrogen, oxygen, sulfur, sulfinyl (—SO—) and sulfonyl (—SO₂—) andis unsubstituted or substituted by lower alkyl or by phenyl-lower alkyl,

R₅, independently of R₂, has one of the meanings mentioned for R₂, and

R₆, independently of R₁, is lower alkoxycarbonyl, or a salt thereof,provided that at least one salt-forming group is present which includesvarious pharmaceutically acceptable acid addition salts thereof.

U.S. Pat. No. 6,087,383 to Singh et al. discloses the bisulfate salt ofthe azapeptide HIV protease inhibitor known as atazanavir which has thestructure

(referred to herein as “atazanavir bisulfate” or “atazanavir sulfate”).

U.S. Patent Publication No. US20050256202A1, published Nov. 17, 2005,discloses processes for preparing the HIV protease inhibitor atazanavirbisulfate and novel forms thereof.

The typical dose of atazanavir to be administered to patients, forexample human beings of approximately 70 kilograms (“kg”) body weight,is from about 3 miligrams (“mg”) to about 1.5 grams (“g”), preferablyfrom about 10 mg to about 1.25 g, for example from about 50 mg to about600 mg per person per day, divided preferably into 1 to 4 single doseswhich may, for example, be of the same size. Usually, children receivehalf of the adult dose.

Ritonavir is typically administered in combination with atazanavirsulfate as another agent having anti-HIV activity. When given as aprotease inhibitor booster, the dosing typically ranges from 100-400 mgtwice daily or, if used as a part of a once-daily regimen, 100-200 mgonce-daily. Ritonavir is commercially available as a prescriptionmedicine from Abbott Laboratories. Abbott Park, Ill., under thetradename Norvir® (ritonavir) for the treatment of HIV.

In preferred embodiments, the polynucleotides and polypeptides of thepresent invention, including allelic and variant forms thereof, haveuses which include, but are not limited to diagnosing individuals toidentify whether a given individual is at a higher risk of developingHIV-1 protease inhibitor-dependent metabolic abnormalities. Anacceptable higher level of a pharmaceutically acceptable dose of a HIV-1protease inhibitor for a patient identified as being at low risk ofdeveloping HIV-1 protease inhibitor-dependent metabolic abnormalitiesmay be 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%,75%, 80%, 85%, 90%, or 95% higher than the prescribed or typical dose,as may be the case.

In another preferred embodiment, the polynucleotides and polypeptides ofthe present invention, including allelic and variant forms thereof, haveuses which include, but are not limited to diagnosing individuals toidentify whether a given individual should be administered acorrespondingly higher dose of a HIV-1 protease inhibitor in order toameliorate an individuals susceptibility or risk for developing HIV-1protease inhibitor-dependent metabolic abnormalities disorder using thegenotype assays of the present invention.

In preferred embodiments, the polynucleotides and polypeptides of thepresent invention, including allelic and variant forms thereof, haveuses which include, but are not limited to use in methods of screeningto identify compounds, particularly HIV-1 protease inhibitor compounds,that have a lower risk of inducing HIV-1 protease inhibitor-dependentmetabolic abnormalities or related disorder in a patient. Suchidentified compounds would be expected to retain all the benefits of aHIV-1 protease inhibitor but would have diminished ability of inducingincreased resistin expression and/or function, or alternatively, capableof increasing resistin expression and/or function to a lesser extentthan a reference compound known to be capable of inducing HIV-1 proteaseinhibitor-dependent metabolic abnormalities. Such compounds would beexpected to be less likely to result in the development of HIV-1protease inhibitor-dependent metabolic abnormalities.

In another embodiment, the polynucleotides and polypeptides of thepresent invention, including allelic and variant forms thereof, eitheralone, or in combination with other polymorphic polynucleotides(haplotypes) are useful as genetic markers for predicting an individualssuspectability to develop HIV-1 protease inhibitor-dependent metabolicabnormalities, and particularly to predicting an individualssuspectability to develop HIV-1 protease inhibitor-dependent metabolicabnormalities upon the administration of an increased dose of a HIV-1protease inhibitor.

Additionally, the polynucleotides and polypeptides of the presentinvention, including allelic and/or variant forms thereof, are usefulfor creating additional antagonists directed against thesepolynucleotides and polypeptides, which include, but are not limited tothe design of antisense RNA, ribozymes, PNAs, recombinant zinc fingerproteins (Wolfe, S A., Ramm, El., Pabo, C O, Structure, Fold, Des., 8(7):739-50, (2000); Kang, J S., Kim, J S, J. Biol, Chem., 275(12):8742-8, (2000); Wang, B S., Pabo, C O, Proc. Natl. Acad. Sci. USA,96 (17):9568-73, (1999); McColl, D J., Honchell, C D., Frankel, A D,Proc. Natl. Acad. Sci. USA, 96 (17):9521-6, (1999); Segal, D J., Dreier,B., Beerli, R R., Barbas, C F—3rd, Proc. Natl. Acad. Sci. USA, 96(6):2758-63, (1999); Wolfe, S A., Greisman, H A., Ramm, E I., Pabo, C O,J. Mol, Biol., 285 (5):1917-34, (1999); Pomerantz, J L., Wolfe, S A.,Pabo, C O, Biochemistry., 37 (4):965-70, (1998); Leon, O., Roth, M.,Biol. Res. 33 (1):21-30 (2000); Berg, J M., Godwin, H A, Ann. Rev.Biophys. Biomol. Struct., 26:357-71 (1997)), in addition to other typesof antagonists which are either described elsewhere herein, or known inthe art.

The polynucleotides and polypeptides of the present invention, includingallelic and/or variant forms thereof, are useful for identifying smallmolecule antagonists directed against the variant forms of thesepolynucleotides and polypeptides, preferably wherein such smallmolecules are useful as therapeutic and/or pharmaceutical compounds forthe treatment, detection, prognosis, and/or prevention of the following,nonlimiting diseases and/or disorders: HIV-1 proteaseinhibitor-dependent metabolic abnormalities, susceptibility todeveloping HIV-1 protease inhibitor-dependent metabolic abnormalitiesupon the administration of a pharmaceutically acceptable amount of anHIV-1 protease inhibitor, susceptibility to developing HIV-1 proteaseinhibitor-dependent metabolic abnormalities upon the administration ofan increased level of a HIV-1 protease inhibitor, adverse reactionsassociated with HIV-1 protease inhibitor, disorders associated withaberrant resistin expression, disorders associated with aberrantresistin regulation, disorders associated with aberrant resistinactivity, disorders associated with aberrant regulation of resistin bycathepsin, disorders associated with aberrant regulation of resistin bycathepsin D, disorders associated with aberrant regulation of resistinby cathepsin E, disorders associated with elevated resistin plasma/serumlevels, HAART-associated lipodystrophy, fat atrophy, fat hypertrophy,dyslipidemia, insulin resistance, cardiovascular disorders, myocardialinfarction, stroke, atherosclerosis, peripheral lipoatrophy, central fataccumulation, hepatic steatosis, lipodystrophy syndrome,lipodystrophy-lie syndromes, hyperglycemia, decreased HDL levels,elevated levels of VLDL, hypertriglyceridemia, hypercholesterolemia, andhyperlipidemia.

Additional disorders which may be detected, diagnosed, identified,treated, prevented, and/or ameliorated by the SNPs and methods of thepresent invention include, the following, non-limiting diseases anddisorders: diabetes, especially Type 2 diabetes, and related diseasessuch as insulin resistance, hyperglycemia, hyperinsulinemia, elevatedblood levels of fatty acids or glycerol, hyperlipidemia, obesity,hypertriglyceridemia, inflammation, Syndrome X, diabetic complications,dysmetabolic syndrome, and related diseases.

Additional uses of the polynucleotides and polypeptides of the presentinvention are provided herein.

Modified Polypeptides and Gene Sequences

The invention further provides variant forms of nucleic acids andcorresponding proteins. The nucleic acids comprise one of the sequencesdescribed in Table I, in which the polymorphic position is occupied byone of the alternative bases for that position. Some nucleic acidsencode full-length variant forms of proteins. Variant genes can beexpressed in an expression vector in which a variant gene is operablylinked to a native or other promoter. Usually, the promoter is aeukaryotic promoter for expression in a mammalian cell. Thetranscription regulation sequences typically include a heterologouspromoter and optionally an enhancer which is recognized by the host. Theselection of an appropriate promoter, for example trp, lac, phagepromoters, glycolytic enzyme promoters and tRNA promoters, depends onthe host selected. Commercially available expression vectors can beused. Vectors can include host-recognized replication systems,amplifiable genes, selectable markers, host sequences useful forinsertion into the host genome, and the like.

The means of introducing the expression construct into a host cellvaries depending upon the particular construction and the target host.Suitable means include fusion, conjugation, transfection, transduction,electroporation or injection, as described in Sambrook, supra. A widevariety of host cells can be employed for expression of the variantgene, both prokaryotic and eukaryotic. Suitable host cells includebacteria such as E. coli, yeast, filamentous fungi, insect cells,mammalian cells, typically immortalized, e.g. , mouse, CHO, human andmonkey cell lines and derivatives thereof. Preferred host cells are ableto process the variant gene product to produce an appropriate maturepolypeptide. Processing includes glycosylation, ubiquitination,disulfide bond formation, general post-translational modification, andthe like. As used herein, “gene product” includes mRNA, peptide andprotein products.

The protein may be isolated by conventional means of proteinbiochemistry and purification to obtain a substantially pure product,i.e., 80, 95 or 99% free of cell component contaminants, as described inJakoby, Methods in Enzymology Volume 104, Academic Press, New York(1984); Scopes, Protein Purification, Principles and Practice, 2ndEdition, Springer-Verlag, New York (1987); and Deutscher (ed), Guide toProtein Purification, Methods in Enzymology, Vol. 182 (1990). If theprotein is secreted, it can be isolated from the supernatant in whichthe host cell is grown. If not secreted, the protein can be isolatedfrom a lysate of the host cells.

Haplotype Based Genetic Analysis

The invention further provides methods of applying the polynucleotidesof the present invention to the elucidation of haplotypes. Suchhaplotypes may be associated with any one or more of the diseaseconditions referenced elsewhere herein. A “haplotype” is defined as thepattern of a set of alleles of single nucleotide polymorphisms along achromosome. For example, consider the case of three single nucleotidepolymorphisms (SNP1, SNP2, and SNP3) in one chromosome region, of whichSNP 1 is an A/G polymorphism, SNP2 is a G/C polymorphism, and SNP3 is anA/C polymorphism. A and G are the alleles for the first, G and C for thesecond and A and C for the third SNP. Given two alleles for each SNP,there are three possible genotypes for individuals at each SNP. Forexample, for the first SNP, A/A, A/G and G/G are the possible genotypesfor individuals. When an individual has a genotype for a SNP in whichthe alleles are not the same, for example A/G for the first SNP, thenthe individual is a heterozygote. When an individual has an A/G genotypeat SNP1, G/C genotype at SNP2, and A/C genotype at SNP3, there are fourpossible combinations of haplotypes (A, B, C, and D) for thisindividual. The set of SNP genotypes of this individual alone would notprovide sufficient information to resolve which combination ofhaplotypes this individual possesses. However, when this individual'sparents' genotypes are available, haplotypes could then be assignedunambiguously. For example, if one parent had an A/A genotype at SNP1, aG/C genotype at SNP2, and an A/A genotype at SNP3, and the other parenthad an A/G genotype at SNP1, C/C genotype at SNP2, and C/C genotype atSNP3, while the child was a heterozygote at all three SNPs, there isonly one possible haplotype combination, assuming there was no crossingover in this region during meiosis.

When the genotype information of relatives is not available, haplotypeassignment can be done using the long range-PCR method (Clark, A. G. MolBiol Evol 7 (2): 111-22 (1990); Clark, A. G., K. M. Weiss, et al. Am JHum Genet 63 (2): 595-612 (1998); Fullerton, S. M., A. G. Clark, et al.,Am J Hum. Genet 67 (4): 881-900 (2000); Templeton, A. R., A. G. Clark,et al., Am J Hum Genet 66 (1): 69-83 (2000)). When the genotyping resultof the SNPs of interest are available from general population samples,the most likely haplotypes can also be assigned using statisticalmethods (Excoffier, L. and M. Slatkin. Mol Biol Evol 12 (5): 921-7(1995); Fallin, D. and N. J. Schork, Am J Hum Genet 67 (4): 947-59(2000); Long, J. C., R. C. Williams, et al., Am J Hum Genet 56 (3):799-810 (1995)).

Once an individual's haplotype in a certain chromosome region (i.e.,locus) has been determined, it can be used as a tool for geneticassociation studies using different methods, which include, for example,haplotype relative risk analysis (Knapp, M., S. A. Seuchter, et al., AmJ Hum Genet 52 (6): 1085-93 (1993); Li, T., M. Arranz, et al., SchizophrRes 32 (2): 87-92 (1998); Matise, T. C., Genet Epidemiol 12 (6): 641-5(1995); Ott, J., Genet Epidemiol 6 (1): 127-30 (1989); Terwilliger, J.D. and J. Ott, Hum Hered 42 (6): 337-46 (1992)). Haplotype based geneticanalysis, using a combination of SNPs, provides increased detectionsensitivity, and hence statistical significance, for geneticassociations of diseases, as compared to analyses using individual SNPsas markers. Multiple SNPs present in a single gene or a continuouschromosomal region are useful for such haplotype-based analyses.

Uses of the Polynucleotides

Each of the polynucleotides identified herein can be used in numerousways as reagents. The following description should be consideredexemplary and utilizes known techniques.

Increased or decreased expression of the gene in affected organisms ascompared to unaffected organisms can be assessed using polynucleotidesof the present invention. Any of these alterations, including alteredexpression, or the presence of at least one SNP of the present inventionwithin the gene, can be used as a diagnostic or prognostic marker.

The invention provides a diagnostic method useful during diagnosis of adisorder, involving measuring the presence or expression level ofpolynucleotides of the present invention in cells or body fluid from anorganism and comparing the measured gene expression level with astandard level of polynucleotide expression level, whereby an increaseor decrease in the gene expression level compared to the standard isindicative of a disorder.

By “measuring the expression level of a polynucleotide of the presentinvention” is intended qualitatively or quantitatively measuring orestimating the level of the polypeptide of the present invention or thelevel of the mRNA encoding the polypeptide in a first biological sampleeither directly (e.g., by determining or estimating absolute proteinlevel or mRNA level) or relatively (e.g., by comparing to thepolypeptide level or mRNA level in a second biological sample).Preferably, the polypeptide level or mRNA level in the first biologicalsample is measured or estimated and compared to a standard polypeptidelevel or mRNA level, the standard being taken from a second biologicalsample obtained from an individual not having the disorder or beingdetermined by averaging levels from a population of organisms not havinga disorder. As will, be appreciated in the art, once a standardpolypeptide level or mRNA level is known, it can be used repeatedly as astandard for comparison.

By “biological sample” is intended any biological sample obtained froman organism, body fluids, cell line, tissue culture, or other sourcewhich contains the polypeptide of the present invention or mRNA. Asindicated, biological samples include body fluids (such as the followingnon-limiting examples, sputum, amniotic fluid, urine, saliva, breastmilk, secretions, interstitial fluid, blood, serum, spinal fluid, etc.)which contain the polypeptide of the present invention, and other tissuesources found to express the polypeptide of the present invention.Methods for obtaining tissue biopsies and body fluids from organisms arewell known in the art. Where the biological sample is to include mRNA, atissue biopsy is the preferred source.

The method(s) provided above may preferably be applied in a diagnosticmethod and/or kits in which polynucleotides and/or polypeptides areattached to a solid support. In one exemplary method, the support may bea “gene chip” or a “biological chip” as described in U.S. Pat. Nos.5,837,832, 5,874,219, and 5,856,174. Further, such a gene chip withpolynucleotides of the present invention attached may be used toidentify polymorphisms between the polynucleotide sequences, withpolynucleotides isolated from a test subject. The knowledge of suchpolymorphisms (i.e. their location, as well as, their existence) wouldbe beneficial in identifying disease loci for many disorders, includingproliferative diseases and conditions. Such a method is described inU.S. Pat. Nos. 5,858,659 and 5,856,104. The U.S. patents referencedsupra are hereby incorporated by reference in their entirety herein.

The present invention encompasses polynucleotides of the presentinvention that are chemically synthesized, or reproduced as peptidenucleic acids (PNA), or according to other methods known in the art. Theuse of PNAs would serve as the preferred form if the polynucleotides areincorporated onto a solid support, or gene chip. For the purposes of thepresent invention, a peptide nucleic acid (PNA) is a polyamide type ofDNA analog and the monomeric units for adenine, guanine, thymine andcytosine are available commercially (Perceptive Biosystems). Certaincomponents of DNA, such as phosphorus, phosphorus oxides, or deoxyribosederivatives, are not present in PNAs. As disclosed by P. E. Nielsen, M.Egholm, R. H. Berg and O. Buchardt, Science 254, 1497 (1991); and M.Egholm, O. Buchardt, L. Christensen, C. Behrens, S. M. Freier, D. A.Driver, R. H. Berg, S. K. Kim, B. Norden, and P. E. Nielsen, Nature 365,666 (1993), PNAs bind specifically and tightly to complementary DNAstrands and are not degraded by nucleases. In fact, PNA binds morestrongly to DNA than DNA, itself does. This is probably because there isno electrostatic repulsion between the two strands, and also thepolyamide backbone is more flexible. Because of this, PNA/DNA duplexesbind under a wider range of stringency conditions than DNA/DNA duplexes,making it easier to perform multiplex hybridization. Smaller probes canbe used than with DNA due to the stronger binding characteristics ofPNA:DNA hybrids. In addition, it is more likely that single basemismatches can be determined with PNA/DNA hybridization because a singlemismatch in a PNA/DNA 15-mer lowers the melting point (T.sub.m) by8°-20° C., vs. 4°-16° C. for the DNA/DNA 15-mer duplex. Also, theabsence of charge groups in PNA means that hybridization can be done atlow ionic strengths and reduce possible interference by salt during theanalysis.

Polynucleotides of the present invention are also useful in genetherapy. One goal of gene therapy is to insert a normal gene into anorganism having a defective gene, in an effort to correct the geneticdefect. The polynucleotides disclosed in the present invention offer ameans of targeting such genetic defects in a highly accurate manner.Another goal is to insert a new gene that was not present in the hostgenome, thereby producing a new trait in the host cell. In one example,polynucleotide sequences of the present invention may be used toconstruct chimeric RNA/DNA oligonucleotides corresponding to saidsequences, specifically designed to induce host cell mismatch repairmechanisms in an organism upon systemic injection, for example(Bartlett, R. J., et al., Nat. Biotech, 18:615-622 (2000), which ishereby incorporated by reference herein in its entirety). Such RNA/DNAoligonucleotides could be designed to correct genetic defects in certainhost strains, and/or to introduce desired phenotypes in the host (e.g.,introduction of a specific polymorphism within an endogenous genecorresponding to a polynucleotide of the present invention that mayameliorate and/or prevent a disease symptom and/or disorder, etc.).Alternatively, the polynucleotide sequence of the present invention maybe used to construct duplex oligonucleotides corresponding to saidsequence, specifically designed to correct genetic defects in certainhost strains, and/or to introduce desired phenotypes into the host(e.g., introduction of a specific polymorphism within an endogenous genecorresponding to a polynucleotide of the present invention that mayameliorate and/or prevent a disease symptom and/or disorder, etc). Suchmethods of using duplex oligonucleotides are known in the art and areencompassed by the present invention (see EP1007712, which is herebyincorporated by reference herein in its entirety).

REFERENCES

1. Carr, A., HIV lipodystrophy: risk factors, pathogenesis, diagnosisand management. Aids, 2003. 17 Suppl 1: p. S141-8.

2. Grinspoon, S. and A. Carr, Cardiovascular risk and body-fatabnormalities in HIV-infected adults. N Engl J Med, 2005. 352(1): p.48-62.

3. Steppan, C. M., et al., The hormone resistin links obesity todiabetes. Nature, 2001. 409(6818): p. 307-12.

4. Rajala, M. W., et al., Adipose-derived resistin and gut-derivedresistin-like molecule-beta selectively impair insulin action on glucoseproduction. J Clin Invest, 2003. 111(2): p. 225-30.

5. Banerjee, R. R., et al., Regulation of fasted blood glucose byresistin. Science, 2004. 303(5661): p. 1195-8.

6. Conneely, K. N., et al., Variation in the resistin gene is associatedwith obesity and insulin-related phenotypes in Finnish subjects.Diabetologia, 2004. 47(10): p. 1782-8.

7. Mattevi, V. S., V. M. Zembrzuski, and M. H. Hutz, A resistin genepolymorphism is associated with body mass index in women. Hum Genet,2004. 115(3): p. 208-12.

8. Ochi, M., et al., The absence of evidence for major effects of thefrequent SNP+299G>A in the resistin gene on susceptibility to insulinresistance syndrome associated with Japanese type 2 diabetes. DiabetesRes Clin Pract, 2003. 61(3): p. 191-8.

9. Kaser, S., et al., Resistin messenger-RNA expression is increased byproinflammatory cytokines in vitro. Biochem Biophys Res Commun, 2003.309(2): p. 286-90.

10. Lehrke, M., et al., An inflammatory cascade leading tohyperresistinemia in humans. PLoS Med, 2004. 1(2): p. e45.

11. Dash, C., et al., Aspartic peptidase inhibitors: implications indrug development. Crit Rev Biochem Mol Biol, 2003. 38(2): p. 89-119.

12. Ohtaka, H., et al., Thermodynamic rules for the design of highaffinity HIV-1 protease inhibitors with adaptability to mutations andhigh selectivity towards unwanted targets. Int J Biochem Cell Biol,2004. 36(9): p. 1787-99.

13. Patel, S. D., et al., Disulfide-dependent multimeric assembly ofresistin family hormones. Science, 2004. 304(5674): p. 1154-8.

14. Gray, W. R., Disulfide structures of highly bridged peptides: a newstrategy for analysis. Protein Sci, 1993. 2(10): p. 1732-48.

15. Chou, K. C., et al., Predicting human immunodeficiency virusprotease cleavage sites in proteins by a discriminant function method.Proteins, 1996. 24(1): p. 51-72.

16. Carr, A., HIV protease inhibitor-related lipodystrophy syndrome.Clin Infect Dis, 2000. 30 Suppl 2: p. S135-42.

EXAMPLES Example 1 Method of Discovering Cathepsin D and Cathepsin EProteolytic Degradation of Resistin

As described elsewhere herein, a SNP was identified in the resistingene, referred to herein as SNP1, that was determined to be associatedwith the incidence of lipodystrophy in patients administered HAARTtherapy. On account of the potential for HIV positive patients to haveelevated plasma/serum levels of resistin, in conjunction with the factthat some aspartyl proteases may be inhibited by HIV-1 proteaseinhibitors, the inventors sought to determine whether either of thelatter could serve as the basis for the observed association of resistinto lipodystrophy.

The protease assays were performed as follows:

HIV-1 Protease Inhibitors: HIV-1 protease inhibitors ritonavir andlopinovir were purified by reverse phase high-performance liquidchromatography from commercial pharmaceutical preparations. Atazanavirwas synthesized by and provided in pure form from the Chemistry Divisionof Bristol-Myers Squibb Co. All compounds were dissolved in 100% DMSO(Sigma). The final concentration of DMSO was 0.2% in experiments thatincluded a protease inhibitor.

Recombinant Proteins: Recombinant human and mouse resistin were obtainedfrom Santa Cruz Biotech. Recombinant cathepsin D and cathepsin E wereobtained from R&D Systems Inc. Recombinant HIV-1 protease was obtainedfrom Protein One. Recombinant renin was obtained from Cayman Chemicaland recombinant trypsin was from Promega.

Alkylation/Reduction of Cysteine Residues: Lyophilized resistin (24micrograms) was resuspended in 50 ul of fresh UA buffer (8 M urea, 0.4 Mammonium bicarbonate) and 5 ul of 45 mM DTT. The mixture was thenincubated at 50° C. for 15 min, followed by addition of 5 ul of 0.1 Miodoacetamide. The reaction was then incubated for an additional 15 minin the dark at room temperature, followed by the addition of 140 ul ofH2O.

Preparation of Proteases: Lyophilized cathepsin D (10 ug) wasresuspended in 100 ul H2O. Cathepsin D was activated prior to use byincubating at 20 ng/ul in CathD buffer (100 mM NaOAc, 200 mM NaCl, pH4.7) for 30 min at 37° C. Lyophilized cathepsin E (10 ug) wasresuspended in 100 ul of reconstitution buffer (25 mM MES, 0.15 M NaCl,pH 6.5). Cathepsin E was activated prior to use by incubating at 20ng/ul in CathE buffer (100 mM NaOAc, 100 mM NaCl, pH 4.7) for 30 min atroom temperature. Renin was diluted to 20 ng/ul in renin buffer (50 mMTris-HCl, pH 8, 100 mM NaCl) prior to use. Lyophilized trypsin (20 ug)was resuspended in 500 ul reconstitution buffer provided by themanufacturer (Promega). Trypsin was activated prior to use by incubatingfor 15 min at 30° C. HIV-1 protease was used directly from themanufacturer.

Protease Digestion Experiments: In all protease digestion reactions, 1ug of alkylated and reduced resistin was incubated with either 1 ugHIV-1 protease, 0.1 ug cathepsin D, 0.1 ug cathepsin E or 0.1 ugtrypsin. HIV-1 protease digestion was performed in HIV-1 PR buffer (50mM MES (pH 6), 100 mM NaCl). Trypsin digestion was performed in TEbuffer (10 mM Tris (pH 8), 0.1 mM EDTA (pH 8)). The respective reactionbuffers for cathepsin D, cathepsin E and renin are listed above under“Preparation of proteases”. For experiments that contained HIV-1protease inhibitors, varying concentrations of atazanavir, ritonavir orlopinovir were added to the protease digestion reactions. Controlexperiments that did not contain protease inhibitor contained additional0.2% DMSO (vehicle control). All protease digestion experiments wereincubated for 24 hr at 37° C. with the exception of experimentscontaining cathepsin E, which were incubated at room temperature.Reactions were quenched in LDS loading dye (Invitrogen) containingreducing agent (Invitrogen). Reactions were subsequently heated at 70°C. for 5 min prior analysis by SDS-PAGE.

SDS-PAGE Analysis: Cleavage products were resolved using 4-12% Bis-TrisNUPAGE® Gels (Invitrogen). Gels were run at 200 V for 20 min in MESbuffer. Protein bands were visualized by staining with Gel Code reagent(Pierce).

Four aspartyl proteases, HIV-1 protease, cathepsin D, cathepsin E andrenin were evaluated for their ability to cleave resistin (MW ca. 10.4kDa) in vitro. As shown in FIG. 1, incubation with HIV-1 protease,cathepsin D and cathepsin E resulted in the appearance of a ca. 7 kDaresistin cleavage product. Additional cleavage products were notdetectable by SDS-PAGE, suggesting that the remaining ca. 3.4 kDafragment was further proteolyzed. Consistent with expectation, resistinwas completely degraded by the non-specific protease, trypsin, but wasrefractory to cleavage by the highly specific protease, renin.

In addition, the protease inhibitors atazanavir and ritonavir were alsoevaluated for their ability to inhibit the proteolysis of resistin bycathepsin D and cathepsin E. As shown in FIG. 2, cleavage of resistin bycathepsin D and cathepsin E was not detectably inhibited by atazanavir,however, cleavage of resistin by cathepsin D and cathepsin E byritonavir at 30 μM and 10 μM, respectively. Cathepsin D and cathepsin Ewere fully inhibited by ritonavir at 100 μM and 30 μM, respectively.

Ritonavir is known in the art to cause metabolic abnormalities, whileatazanavir is not. The above results support the hypothesis thatscreening for HIV-1 protease inhibitor compound that lack the ability toinhibit cathepsin D and/or cathepsin E-dependent proteolysis of resistinwould be desirable in order to identify compounds that have a decreasedlikelihood of causing metabolic abnormalities in patients.

Example 2 Method of Discovering the Single Nucleotide Polymorphisms(SNPs) of the Present Invention

Elevated lipid levels, insulin resistance and changes in body fat,collectively known as lipodystrophy, are common in HIV infectedindividuals (Grinspoon, S. & Carr, A., N. Engl. J. Med. 352: 48-62(2005)). Highly active anti-retroviral therapy (HAART) is one of thestrongest predictors of lipodystrophy (Grinspoon, S. & Carr, A., N.Engl. J. Med. 352: 48-62 (2005)). With the reduction in mortalityresulting from treatment with HAART, these metabolic side-effects are ofconcern as they are well-established risk factors for cardiovasculardisease. The mechanistic basis of HAART-associated lipodystrophy ispoorly understood, however.

Metabolic profiles of participants in a HAART-trial were clustered toidentify a sub-group of patients who had a normal metabolic profile atbaseline but developed significantly elevated plasma lipid levels andinsulin resistance on HAART. 189 participants of a HAART trial (Dube, M.P. et al., AIDS 19: 1807-18 (2005)) who had metabolic measurements atbaseline and up to 64 weeks of treatment were analyzed. All subjectsgave written informed consent. The metabolic profile for each patientafter 32 weeks of HAART comprised measurements of body mass index,total, LDL, HDL and non-HDL cholesterol, triglycerides, glucose andinsulin resistance by homeostasis model adjustment (HOMA-IR).Individuals with similar metabolic profiles were grouped together, andthis process was iteratively repeated until all individuals wereclustered into groups. By using change in information content as afunction of cluster number, the optimal number of clusters wasdetermined, which in this case was two. Examination of mean values ofmetabolic traits led to labeling one cluster normal (N=142) and theother high-risk (N=47).

The 189 individuals in these two clusters were genotyped for 285 SNPs in135 candidate genes. Genes were selected based on their likelyinvolvement in regulating lipid and glucose metabolism, cytokines, drugmetabolizing enzymes and transcription factors that regulate expressionof these genes. Genes were also selected based upon those genes that hadsignificantly perturbed expression patterns in cell-culture afterexposure to protease inhibitors. Single nucleotide polymorphisms (SNPs)that were more likely to affect the activity (e.g. coding sequencechanges) or expression (promoter or near splice-sites) of the gene wereselected for further analysis.

Example 3 Method of Genotyping Each SNP of the Present Invention

Genomic DNA samples from patients enrolled in a HAART trial (Dube, M. P.et al., AIDS 19: 1807-18 (2005)) were genotyped for 285 SNPs identifiedin 135 candidate genes (see Example 1) and evaluated for associationwith HAART-dependent metabolic abnormalities.

All analyses were based on data collected at baseline and up to 64 weeksof treatment. DNA was extracted from frozen blood by a third-party.

Genotyping was performed using the 5′ nuclease assay, essentially asdescribed (Ranade K et al., Genome Research 11: 1262-1268 (2001); whichis hereby incorporated by reference herein in its entirety), with thefollowing modifications: six nanograms of genomic DNA were used in a 8ul reaction. All PCR reactions were performed in an ABI 9700 machine andfluorescence was measured using an ABI 7900 machine.

Genotyping of the SNPs of the present invention was performed using setsof TAQMAN® probes (100 uM each) and primers (100 uM each) specific toeach SNP. Each probe/primer set was manually designed using ABI PrimerExpress software (Applied Biosystems). Genomic samples were prepared asdescribed herein. The following TAQMAN® probes and primers were utilizedfor one of the resistin SNPs. Genotyping primers for the other 284 SNPshave not been provided.

TAQMAN ® TAQMAN ® Reference Variable Forward Reverse TAQMAN ® TAQMAN ®SNP Primer Primer Probe Probe SNP1 CCGGCTCCCTA GAGTCAGGTCTGTCCCCAAGGGTCT TCCCCAAGGGTCT AGTGAGGAC TGCCAGGG C AGAGACCTCAC TAGAGACCTCACT (SEQ ID NO: 3) (SEQ ID NO: 4) (SEQ ID NO: 5) G (SEQ ID NO:6) **The allelic nucleotide in each probe sequence is shown in bold andunderlined.

The genotype assay conditions are provided below.

Components: Final Concentration: 2x PE Master Mix (#4318157) 1X 100 uMFAM labeled probe 200 nmol 100 uM VIC labeled probe 200 nmol Forward PCRprimer 600 nmol Reverse PCR primer 600 nmol 6 ng template DNA asrequired ddH20 volume to 8 ul

TAQMAN® thermo-cycling was performed on Perkin Elmer PE 9700 machinesusing the following cycling conditions below:

1) 50 C for 2 minutes

2) 95 C for 10 seconds*

3) 94 C for 15 seconds

4) 62 C for 1 minute

5) 4 C hold

* Steps 2-4 were cycled 40 times

Analysis of genotypes was performed by using the Applied Biosystems ABI7900 HT sequence detection system.

Example 4 Statistical Analysis of the Association BetweenHAART-Dependent Metabolic Abnormalities and the SNPs of the PresentInvention

The association between HAART-dependent metabolic abnormalities and thesingle nucleotide polymorphisms of the present invention wereinvestigated by applying statistical analysis to the results of thegenotyping assays described elsewhere herein. The central hypothesis ofthis analysis is that a predisposition to develop HIV-1 proteaseinhibitor-dependent metabolic abnormalities may be conferred by specificgenomic factors. The analysis attempted to identify one or more of thesefactors in genomic DNA samples from index cases and matched controlsubjects who were exposed to HAART in a clinical study (see Example 2).

SNPs of the present invention were examined for association with HIV-1protease inhibitor-dependent metabolic abnormalities using 3(genotypes)×2 (high risk and low risk cluster) contingency tables.

Methods

Sample: Investigators in the BMS clinical trials diagnosed HIV-1protease inhibitor-dependent metabolic abnormalities in some subjects.

Measures: Single nucleotide polymorphisms (SNPs) in human resistin weregenotyped on all subjects essentially as described in Example 2 herein.The SNPs that were genotyped likely represent a sample of thepolymorphic variation in each gene and are not exhaustive with regard tocoverage of the total genetic variation that may be present in eachgene. The SNP for which a statistical association to HIV-1 proteaseinhibitor-dependent metabolic abnormalities was confirmed is provided asSNP1.

Statistical Analyses: All statistical analyses were done using SPSS®version 12 (Chicago, Ill., US).

Clustering: Traits measured after 32 weeks of HAART were standardized tohave a mean of 0 and variance of 1 and then used in the clustering. Foreach individual a profile was made of body mass index, total, LDL, HDLand non-HDL cholesterol, triglyceride, glucose, and HOMA-IR. Theseprofiles were clustered together using “two-step” clustering to identifysub-groups with more homogeneous metabolic profiles. In this clusteringmethod, data are a first grouped into sub-clusters using agglomerativeclustering and then cluster assignment is refined to determine theoptimal number of clusters. The optimal number of clusters wasdetermined in two steps. First, Bayesian information criterion wascalculated for the specified number of clusters to obtain an initialestimate of cluster number. Second, this estimate was refined by findingthe greatest change in distance between two clusters in each stage ofhierarchical clustering The distance between two clusters was defined asthe decrease in log-likelihood resulting from the two clusters beingcombined into a single cluster. Up to 20 clusters were permitted, butthe algorithm repeatedly determined that a two cluster solution wasoptimal. To assess stability of the cluster solution, multiple runs(N=20) of clustering were performed on different days using randomlysorted data. All runs produced clusters identical to those describedearlier. Differences in means between clusters for metabolic traits andbody fat were evaluated using Kruskal-Wallis or repeated measures ANOVA.

Genetic association between SNPs and clusters was assessed using χ² orFisher's exact tests.

A C/T SNP thirty-nine base-pairs downstream of the second exon of theresistin gene was the most significantly associated with clustermembership (P=0.0003). The frequency of this SNP in the normal clusterwas 0.16 and it was 0.33 in the high-risk cluster. C/T heterozygotes andT/T homozygotes were 2.7 (95% C.I. 1.3-5.3) and 19 (95% C.I. 2-183)times more likely to be classified in the high-risk cluster thanwild-type (P=0.001).

The nucleotide sequence of the resistin gene containing the referenceallele (“C”) for SNP1 at nucleotide 1398 is provided in FIGS. 3A-B (SEQID NO:1); while the nucleotide sequence of the resistin gene containingthe variable allele (“T”) for SNP1 at nucleotide 1398 is provided inFIGS. 4A-B (SEQ ID NO:2).

Resistin was cloned as a hormone that links obesity and insulinresistance (Steppan, C. M. et al., Nature 409: 307-12 (2001). Increasedresistin resulted in mice with abnormal glucose tolerance (Steppan, C.M. et al., Nature 409: 307-12 (2001). Conversely, resistin knockout micehad lower fasting blood glucose (Banerjee, R. et al., Science 203:1195-98 (2004)). Consistent with the genetic association observed inthis study, SNPs in resistin have been variably associated withmetabolic traits captured by the clusters described above including bodymass index, fat mass, insulin resistance and diabetes (Engert, J. C., etal., Diabetes 51: 1629-34 (2002); Engert, J. C., et al. Diabetes 51:1629-34 (2002); Wang, H., et al., J. Clin. Endo. Metab. 87: 2520-24(2002); Sentinelli, F. et al., Diabetes 51: 860-62 (2002); Coneely, K.N. et al., Diabetologia 47: 1782-88 (2004); and Osawa, H. et al., Am. J.Hum. Genet. 75: 678-86 (2004).

These results suggest that polymorphisms in the resistin genecontributes to differences in susceptibility to HIV-1 proteaseinhibitor-dependent metabolic abnormalities independent of othersignificant predictors such as age, sex and body mass index.

The utility, in general, of each of these significant SNP-HIV-1 proteaseinhibitor-dependent metabolic abnormalities event associations is thatthey suggest (1) such SNPs may be causally involved, alone or incombination with other SNPs in the respective gene regions withsusceptibility to HIV-1 protease inhibitor-dependent metabolicabnormalities events resulting from exposure to a HIV-1 proteaseinhibitor; (2) such SNPs, if not directly causally involved, arereflective of an association because of linkage disequilibrium with oneor more other SNPs that may be causally involved, alone or incombination with other SNPs in the respective gene regions withsusceptibility to HIV-1 protease inhibitor-dependent metabolicabnormalities resulting from exposure to a HIV-1 protease inhibitor; (3)such SNPs may be useful in establishing haplotypes that may be used tonarrow the search for and identify polymorphisms or combinations ofpolymorphisms that may be causally, alone or in combination with otherSNPs in the respective gene regions with susceptibility to HIV-1protease inhibitor-dependent metabolic abnormalities resulting fromexposure to a HIV-1 protease inhibitor; and (4) such SNPs, if used toestablish haplotypes that are identified as causally involved in suchevent susceptibility, may be used to predict which subjects are mostlikely to experience such events when exposed to a HIV-1 proteaseinhibitor-dependent metabolic abnormalities resulting from exposure to aHIV-1 protease inhibitor. The term “respective gene regions” shall beconstrued to refer to those regions of each gene which have been used toidentify the SNPs of the present invention.

Example 5 Method of Isolating the Native Forms of the Human ResistinGene

A number of methods have been described in the art that may be utilizedin isolating the native forms of the human resistin gene. Specificmethods are referenced below and are hereby incorporated by referenceherein in their entireties. The artisan, skilled in the molecularbiology arts, would be able to isolate the native form of human resistinbased upon the methods and information contained, and/or referenced,therein.

1. Human Resistin (gi|NM_(—)020415; SEQ ID NO:7; chr19:7638972-7641340dbSNP ID rs3219177).

2. Adeghate, E., Cell. Mol. Life Sci. 61 (19-20), 2485-2496 (2004).

3. Azuma, K., et al., Horm. Metab. Res. 36 (8), 564-570 (2004).

4. Cho, Y. M., et al., Diabetologia 47 (3), 559-565 (2004).

5. Motojima, K., J. Endocrinol. Invest. 26 (12), 1171-1173 (2003).

6. Seo, J. B., et al., Mol. Endocrinol. 17 (8), 1522-1533 (2003).

7. Smith, S. R., et al., Diabetes 52 (7), 1611-1618 (2003).

8. Banerjee, R. R. and Lazar, M. A., J. Mol. Med. 81 (4), 218-226(2003).

9. Tan, M. S., et al., J. Clin. Endocrinol. Metab. 88 (3), 1258-1263(2003).

10. Ma, X., et al., J. Clin. Endocrinol. Metab. 87 (9), 4407-4410(2002).

11. Pizzuti, A., et al., J. Clin. Endocrinol. Metab. 87 (9), 4403-4406(2002).

12. Wang, H., et al., J. Clin. Endocrinol. Metab. 87 (6), 2520-2524(2002).

13. McTeman, P. G., et al., J. Clin. Endocrinol. Metab. 87 (5), 2407(2002).

14. Steppan, C. M., et al., Proc. Natl. Acad. Sci. U.S.A. 98 (2),502-506 (2001).

15. Holcomb, I. N., et al., EMBO J. 19 (15), 4046-4055 (2000).

16. Bennett, M. and Reed, R., Science 262 (5130), 105-108 (1993).

Methods of isolation for the human resistin gene of the presentinvention may also be found in reference to the references cited in theGENBANK® accession nos. for each gene provided herein which are herebyincorporated by reference herein.

Example 6 Method of Isolating the Polymorphic Forms of the HumanResistin Gene of the Present Invention

Since the allelic genes of the present invention represent genes presentwithin at least a subset of the human population, these genes may beisolated using the methods provided in Example 4 above. For example, thesource DNA used to isolate the allelic gene may be obtained through arandom sampling of the human population and repeated until the allelicform of the gene is obtained. Preferably, random samples of source DNAfrom the human population are screened using the SNPs and methods of thepresent invention to identify those sources that comprise the allelicform of the gene. Once identified, such a source may be used to isolatethe allelic form of the gene(s). The invention encompasses the isolationof such allelic genes from both genomic and/or cDNA libraries createdfrom such source(s).

In reference to the specific methods provided in Example 4 above, it isexpected that isolating the polymorphic alleles of the human resistingene would be within the skill of an artisan trained in the molecularbiology arts. Nonetheless, a detailed exemplary method of isolating atleast one of the resistin polymorphic alleles, in this case the variantform of SNP31(“T” nucleotide at 1398 of SEQ ID NO:1) is provided.Briefly,

First, the individuals with the “t” allele at the locus corresponding tonucleotide 1398 of SEQ ID NO:1 or 2 are identified by genotyping thegenomic DNA samples using the method outlined in Example 2 herein. Othermethods of genotyping may be employed, such as the FP-SBE method (Chenet al., Genome Res., 9(5):492-498 (1999)), or other methods describedherein. DNA samples publicly available (e.g., from the Coriell Institute(Collingswood, N.J.) or from the clinical samples described herein maybe used. Oligonucleotide primers that are used for this genotyping assayare provided in Example 2.

By analyzing genomic DNA samples, individuals with the C1398T form ofthe SNP1 variant may be identified. Once identified, clones comprisingthe genomic sequence may be obtained using methods well known in the art(see Sambrook, J., E. F. Fritsch, and T. Maniatis, 1989, MolecularCloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y.; and Current Protocols in Molecular Biology, 1995,F. M. Ausubel et al., eds., John Wiley and Sons, Inc., which are herebyincorporated by reference herein.).

If cDNA clones of the coding sequence of this allele of the gene are ofinterest, such clones may be obtained in accordance with the followingsteps. Next, Lymphoblastoid cell lines may be obtained from the CoriellInstitute. These cells can be grown in RPMI-1640 medium with L-glutamineplus 10% FCS at 37degrees. PolyA+RNA are then isolated from these cellsusing OLIGOTEX® Direct Kit (Life Technologies).

First strand cDNA (complementary DNA) is produced using SUPERSCRIPT®Preamplification System for First Strand cDNA Synthesis (LifeTechnologies, Cat No 18089-011) using these polyA+RNA as templates, asspecified in the users manual which is hereby incorporated herein byreference in its entirety. Specific cDNA encoding the human resistinprotein is amplified by polymerase chain reaction (PCR) using a forwardprimer which hybridizes to the 5′-UTR region, a reverse primer whichhybridizes to the 3′-UTR region, and these first strand cDNA astemplates (Sambrook, Fritsch et al. 1989). Alternatively, these primersmay be designed using Primer3 program (Rozen S 2000). Restriction enzymesites (example: SalI for the forward primer, and NotI for reverseprimer) are added to the 5′-end of these primer sequences to facilitatecloning into expression vectors after PCR amplification. PCRamplification may be performed essentially as described in the owner'smanual of the Expand Long Template PCR System (Roche MolecularBiochemicals) following manufacturer's standard protocol, which ishereby incorporated herein by reference in its entirety.

PCR amplification products are digested with restriction enzymes (suchas SalI and NotI, for example) and ligated with expression vector DNAcut with the same set of restriction enzymes. pSPORT (Invitrogen) is oneexample of such an expression vector. After ligated DNA is introducedinto E. coli cells (Sambrook, Fritsch et al. 1989), plasmid DNA isisolated from these bacterial cells. This plasmid DNA is sequenced toconfirm the presence an intact (full-length) coding region of the humanresistin protein with the variation, if the variation results in changesin the encoded amino acid sequence, using methods well known in the artand described elsewhere herein.

The skilled artisan would appreciate that the above method may beapplied to isolating the other novel human resistin genes of the presentinvention through the simple substitution of applicable PCR andsequencing primers. Such primers may be selected from any one of theapplicable primers provided in herein, or may be designed using thePrimer3 program (Rozen S 2000) as described. Such primers may preferablycomprise at least a portion of any one of the polynucleotide sequencesof the present invention.

Example 7 Method of Engineering the Allelic Forms of the Human ResistinGene of the Present Invention

Aside from isolating the allelic genes of the present invention from DNAsamples obtained from the human population, as described in Example 5above, the invention also encompasses methods of engineering the allelicgenes of the present invention through the application of site-directedmutagenesis to the isolated native forms of the genes. Such methodologycould be applied to synthesize allelic forms of the genes comprising atleast one, or more, of the encoding SNPs of the present invention (e.g.,silent, missense)—preferably at least 1, 2, 3, or 4 encoding SNPs foreach gene.

In reference to the specific methods provided in Example 5 above, it isexpected that isolating the novel polymorphic resistin genes of thepresent invention would be within the skill of an artisan trained in themolecular biology arts. Nonetheless, a detailed exemplary method ofengineering at least one of the resistin polymorphic alleles to comprisethe encoding and/or non-coding polymorphic nucleic acid sequence, inthis case the variant form (C1398T) of SNP1 (SEQ ID NO:2) is provided.Briefly, cDNA clones encoding the human resistin protein may beidentified by homology searches with the BLASTN program (Altschul S F1990) against the GENBANK® non-redundant nucleptide sequence databaseusing the published human resistin cDNA sequence (GENBANK® AccessionNo.: NM_(—)020415). Alternatively, the genomic sequence of the humanresistin gene may be obtained as described herein. After obtaining theseclones, they are sequenced to confirm the validity of the DNA sequences.

However, in the case of the variant form (C1398T) of SNP1, genomicclones would need to be obtained and may be identified by homologysearches with the BLASTN program (Altschul SF 1990) against the GENBANK®non-redundant nucleotide sequence database using the published humanresistin genomic sequence (GENBANK® Accession No.: AC008963.9,nucleotides chr19:7638972-7641340 dbSNP ID rs3219177). Alternatively,the genomic sequence of the human resistin gene may be obtained asdescribed herein. After obtaining these clones, they are sequenced toconfirm the validity of the DNA sequences.

Once these clones are confirmed to contain the intact wild type cDNA orgenomic sequence of the human resistin coding and/or non-coding region,the C 1398T polymorphism (mutation) may be introduced into the nativesequence using PCR directed in vitro mutagenesis (Cormack, B., DirectedMutagenesis Using the Polymerase Chain Reaction. Current Protocols inMolecular Biology, John Wiley & Sons, Inc. Supplement 37: 8.5.1-8.5.10,(2000)). In this method, synthetic oligonucleotides are designed toincorporate a point mutation at one end of an amplified fragment.Following PCR, the amplified fragments are made blunt-ended by treatmentwith Klenow Fragment. These fragments are then ligated and subclonedinto a vector to facilitate sequence analysis. This method consists ofthe following steps.

1. Subcloning of cDNA or genomic insert into a plasmid vector, or BACsequence if the clone is a genomic sequence, containing multiple cloningsites and M13 flanking sequences, such as pUC19 (Sambrook, Fritsch etal. 1989), in the forward orientation. The skilled artisan wouldappreciate that other plasmids could be equally substituted, and may bedesirable in certain circumstances.

2. Introduction of a mutation by PCR amplification of the genomic regiondownstream of the mutation site using a primer including the mutation.(FIG. 8.5.2 in Cormack 2000)). In the case of introducing the C1398Tmutation into the human resistin genomic sequence, the following twoprimers may be used.

M13 reverse sequencing primer: (SEQ ID NO: 12)5′-AGCGGATAACAATTTCACACAGGA-3′. Mutation primer: (SEQ ID NO: 10)5′-GGCAAGCTCCCCAAGGGTCT T AGAGACCTCACTGATCCC-3′

Mutation primer contains the mutation (C1398T) at the 5′ end (in boldand underlined) and a portion of its flanking sequence. M13 reversesequencing primer hybridizes to the pUC19 vector. Subcdoned cDNA orgenomic clone comprising the human resistin cDNA or genomic sequence isused as a template (described in Step 1). A 100 ul PCR reaction mixtureis prepared using 10 ng of the template DNA, 200 uM 4dNTPs, 1 uMprimers, 0.25U Taq DNA polymerase (PE), and standard Taq DNA polymerasebuffer. Typical PCR cycling condition are as follows:

20-25 cycles: 45 sec, 93 degrees  2 min, 50 degrees  2 min, 72 degrees 1cycle: 10 min, 72 degrees

After the final extension step of PCR, 5U Klenow Fragment is added andincubated for 15 min at 30 degrees. The PCR product is then digestedwith the restriction enzyme, EcoRI.

3. PCR amplification of the upstream region is then performed, usingsubcloned cDNA or genomic clone as a template (the product of Step 1).This PCR is done using the following two primers:

M13 forward sequencing primer: (SEQ ID NO: 8)5′-CGCCAGGGTTTTCCCAGTCACGAC-3′. Flanking primer: (SEQ ID NO: 11)5′-GGGATCAGTGAGGTCTCT A AGACCCTTGGGGAGCTTGCC-3′.

Flanking primer is complimentary to the upstream flanking sequence andmutation locus of the C1398T mutation (in bold and underlined). M13forward sequencing primer hybridizes to the pUC19 vector. PCR conditionsand Klenow treatments follow the same procedures as provided in Step 2,above. The PCR product is then digested with the restriction enzyme,HindIII.

4. Prepare the pUC19 vector for cloning the cDNA or genomic clonecomprising the polymorphic locus. Digest pUC19 plasmid DNA with EcoRIand HindII. The resulting digested vector fragment may then be purifiedusing techniques well known in the art, such as gel purification, forexample.

5. Combine the products from Step 2 (PCR product containing mutation),Step 3 (PCR product containing the upstream region), and Step 4(digested vector), and ligate them together using standard blunt-endligation conditions (Sambrook, Fritsch et al. 1989).

6. Transform the resulting recombinant plasmid from Step 5 into E. colicompetent cells using methods known in the art, such as, for example,the transformation methods described in Sambrook, Fritsch et al. 1989.

7. Analyze the amplified fragment portion of the plasmid DNA by DNAsequencing to confirm the point mutation, and absence of any othermutations introduced during PCR. The method of sequencing the insertDNA, including the primers utilized, are described herein or areotherwise known in the art.

Example 8 Alternative Methods of Genotyping Polymorphisms Encompassed bythe Present Invention Preparation of Samples

Polymorphisms are detected in a target nucleic acid from an individualbeing analyzed. For assay of genomic DNA, virtually any biologicalsample (other than pure red blood cells) is suitable. For example,convenient tissue samples include whole blood, semen, saliva, tears,urine, fecal material, sweat, buccal, skin and hair. For assay of cDNAor mRNA, the tissue sample must be obtained from an organ in which thetarget nucleic acid is expressed. For example, if the target nucleicacid is a cytochrome P450, the liver is a suitable source.

Many of the methods described below require amplification of DNA fromtarget samples. This can be accomplished by e.g., PCR. See generally PCRTechnology: Principles and Applications for DNA Amplification (ed. H. A.Erlich, Freeman Press, NY, N.Y., 1992); PCR Protocols: A Guide toMethods and Applications (eds. Innis, et al., Academic Press, San Diego,Calif., 1990); Mattila et al., Nucleic Acids Res. 19, 4967 (1991);Eckert et al., PCR Methods and Applications 1, (1991); PCR (eds.McPherson et al., IRL Press, Oxford); and U.S. Pat. No. 4,683,202.

Other suitable amplification methods include the ligase chain reaction(LCR) (see Wu and Wallace, Genomics 4:560 (1989), Landegren et al.,Science 241:1077 (1988), transcription amplification (Kwoh et al., Proc.Natl. Acad. Sci. USA 86, 1173 (1989), and self-sustained sequencereplication (Guatelli et al., Proc. Nat. Acad. Sci. USA, 87:1874 (1990))and nucleic acid based sequence amplification (NASBA). The latter twoamplification methods involve isothermal reactions based on isothermaltranscription, which produce both single stranded RNA (ssRNA) and doublestranded DNA (dsDNA) as the amplification products in a ratio of about30 or 100 to 1, respectively.

Additional methods of amplification are known in the art or aredescribed elsewhere herein.

Detection of Polymorphisms in Target DNA

There are two distinct types of analysis of target DNA for detectingpolymorphisms. The first type of analysis, sometimes referred to as denovo characterization, is carried out to identify polymorphic sites notpreviously characterized (i.e., to identify new polymorphisms). Thisanalysis compares target sequences in different individuals ,to identifypoints of variation, i.e., polymorphic sites. By analyzing groups ofindividuals representing the greatest ethnic diversity among humans andgreatest breed and species variety in plants and animals, patternscharacteristic of the most common alleles/haplotypes of the locus can beidentified, and the frequencies of such alleles/haplotypes in thepopulation can be determined. Additional allelic frequencies can bedetermined for subpopulations characterized by criteria such asgeography, race, or gender. The de novo identification of polymorphismsof the invention is described in the Examples section.

The second type of analysis determines which form(s) of a characterized(known) polymorphism are present in individuals under test. Additionalmethods of analysis are known in the art or are described elsewhereherein.

Allele-Specific Probes

The design and use of allele-specific probes for analyzing polymorphismsis described by e.g., Saiki et al., Nature 324,163-166 (1986);Dattagupta, EP 235,726, Saiki, WO 89/11548. Allele-specific probes canbe designed that hybridize to a segment of target DNA from oneindividual but do not hybridize to the corresponding segment fromanother individual due to the presence of different polymorphic forms inthe respective segments from the two individuals. Hybridizationconditions should be sufficiently stringent that there is a significantdifference in hybridization intensity between alleles, and preferably anessentially binary response, whereby a probe hybridizes to only one ofthe alleles. Some probes are designed to hybridize to a segment oftarget DNA such that the polymorphic locus aligns with a centralposition (e.g., in a 15-mer at the 7 position; in a 16-mer, at eitherthe 8 or 9 position) of the probe. This design of probe achieves gooddiscrimination in hybridization between different allelic forms.

Allele-specific probes are often used in pairs, one member of a pairshowing a perfect match to a reference form of a target sequence and theother member showing a perfect match to a variant form. Several pairs ofprobes can then be immobilized on the same support for simultaneousanalysis of multiple polymorphisms within the same target sequence.

Tiling Arrays

The polymorphisms can also be identified by hybridization to nucleicacid arrays, some examples of which are described in WO 95/11995. Thesame arrays or different arrays can be used for analysis ofcharacterized polymorphisms. WO 95/11995 also describes sub arrays thatare optimized for detection of a variant form of a precharacterizedpolymorphism. Such a sub array contains probes designed to becomplementary to a second reference sequence, which is an allelicvariant of the first reference sequence. The second group of probes isdesigned by the same principles as described, except that the probesexhibit complementarity to the second reference sequence. The inclusionof a second group (or further groups) can be particularly useful foranalyzing short subsequences of the primary reference sequence in whichmultiple mutations are expected to occur within a short distancecommensurate with the length of the probes (e.g., two or more mutationswithin 9 to bases).

Allele-Specific Primers

An allele-specific primer hybridizes to a site on target DNA overlappinga polymorphism and only primes amplification of an allelic form to whichthe primer exhibits perfect complementarity. See Gibbs, Nucleic AcidRes. 17,2427-2448 (1989). This primer is used in conjunction with asecond primer which hybridizes at a distal site. Amplification proceedsfrom the two primers, resulting in a detectable product which indicatesthe particular allelic form is present. A control is usually performedwith a second pair of primers, one of which shows a single base mismatchat the polymorphic locus and the other of which exhibits perfectcomplementarity to a distal site. The single-base mismatch preventsamplification and no detectable product is formed. The method works bestwhen the mismatch is included in the 3′-most position of theoligonucleotide aligned with the polymorphism because this position ismost destabilizing elongation from the primer (see, e.g., WO 93/22456).

Direct-Sequencing

The direct analysis of the sequence of polymorphisms of the presentinvention can be accomplished using either the dideoxy chain terminationmethod or the Maxam-Gilbert method (see Sambrook et al., MolecularCloning, A Laboratory Manual (2nd Ed., CSHP, New York 1989); Zyskind etal., Recombinant DNA Laboratory Manual, (Acad. Press, 1988)).

Denaturing Gradient Gel Electrophoresis

Amplification products generated using the polymerase chain reaction canbe analyzed by the use of denaturing gradient gel electrophoresis.Different alleles can be identified based on the differentsequence-dependent melting properties and electrophoretic migration ofDNA in solution. Erlich, ed., PCR Technology. Principles andApplications for DNA Amplification, (W .H. Freeman and Co, New York,1992), Chapter 7.

Single-Strand Conformation Polymorphism Analysis

Alleles of target sequences can be differentiated using single-strandconformation polymorphism analysis, which identifies base differences byalteration in electrophoretic migration of single stranded PCR products,as described in Orita et al., Proc. Nat. Acad. Sci. 86,2766-2770 (1989).Amplified PCR products can be generated as described above, and heatedor otherwise denatured, to form single stranded amplification products.Single-stranded nucleic acids may refold or form secondary structureswhich are partially dependent on the base sequence. The differentelectrophoretic mobilities of single-stranded amplification products canbe related to base-sequence differences between alleles of targetsequences.

Single Base Extension

An alternative method for identifying and analyzing polymorphisms isbased on single-base extension (SBE) of a fluorescently-labeled primercoupled with fluorescence resonance energy transfer (FRET) between thelabel of the added base and the label of the primer. Typically, themethod, such as that described by Chen et al., (PNAS 94:10756-61 (1997),uses a locus-specific oligonucleotide primer labeled on the 5′ terminuswith 5-carboxyfluorescein (F AM). This labeled primer is designed sothat the 3′ end is immediately adjacent to the polymorphic locus ofinterest. The labeled primer is hybridized to the locus, and single baseextension of the labeled primer is performed with fluorescently-labeleddideoxyribonucleotides (ddNTPs) in dye-terminator sequencing fashion. Anincrease in fluorescence of the added ddNTP in response to excitation atthe wavelength of the labeled primer is used to infer the identity ofthe added nucleotide.

Example 9 Additional Methods of Genotyping the SNPs of the PresentInvention

The skilled artisan would acknowledge that there are a number of methodsthat may be employed for genotyping a SNP of the present invention,aside from the preferred methods described herein. The present inventionencompasses the following non-limiting types of genotype assays:PCR-free genotyping methods, Single-step homogeneous methods,Homogeneous detection with fluorescence polarization, Pyrosequencing,“Tag” based DNA chip system, Bead-based methods, fluorescent dyechemistry, Mass spectrometry based genotyping assays, TAQMAN® genotypeassays, Invader genotype assays, and microfluidic genotype assays, amongothers.

Specifically encompassed by the present invention are the following,non-limiting genotyping methods: Landegren, U., Nilsson, M. & Kwok, P.Genome Res 8, 769-776 (1998); Kwok, P., Pharmacogenomics 1, 95-100(2000); Gut, I., Hum Mutat 17, 475-492 (2001); Whitcombe, D., Newton, C.& Little, S., Curr Opin Biotechnol 9, 602-608 (1998); Tillib, S. &Mirzabekov, A., Curr Opin Biotechnol 12, 53-58 (2001); Winzeler, E. etal., Science 281, 1194-1197 (1998); Lyamichev, V. et al., Nat Biotechnol17, 292-296 (1999); Hall, J. et al., Proc Natl Acad Sci USA 97,8272-8277 (2000); Mein, C. et al., Genome Res 10, 333-343 (2000);Ohnishi, Y. et al., J Hum Genet 46, 471-477 (2001); Nilsson, M. et al.,Science 265, 2085-2088 (1994); Baner, J., Nilsson, M., Mendel-Hartvig,M. & Landegren, U., Nucleic Acids Res 26, 5073-5078 (1998); Baner, J. etal., Curr Opin Biotechnol 12, 11-15 (2001); Hatch, A., Sano, T., Misasi,J. & Smith, C., Genet Anal 15, 35-40 (1999); Lizardi, P. et al., NatGenet 19, 225-232 (1998); Zhong, X., Lizardi, P.,Huang, X., Bray-Ward,P. & Ward, D., Proc Natl Acad Sci USA 98, 3940-3945 (2001); Faruqi, F.et al. BMC Genomics 2, 4 (2001); Livak, K., Genet Anal 14, 143-149(1999); Marras, S., Kramer, F. & Tyagi, S., Genet Anal 14, 151-156(1999); Ranade, K. et al., Genome Res 11, 1262-1268 (2001); Myakishev,M., Khripin, Y., Hu, S. & Hamer, D., Genome Res 11, 163-169 (2001);Beaudet, L., Bedard, J., Breton, B., Mercuri, R. & Budarf, M., GenomeRes 11, 600-608 (2001); Chen, X., Levine, L. & PY, K., Genome Res 9,492-498 (1999); Gibson, N. et al., Clin Chem 43, 1336-1341 (1997);Latif, S., Bauer-Sardina, I., Ranade, K., Livak, K. & PY, K., Genome Res11, 436-440 (2001); Hsu, T., Law, S., Duan, S., Neri, B. & Kwok, P.,Clin Chem 47, 1373-1377 (2001); Alderborn, A., Kristofferson, A. &Hammerling, U., Genome Res 10, 1249-1258 (2000); Ronaghi, M., Uhlen, M.& Nyren, P., Science 281, 363, 365 (1998); Ronaghi, M., Genome Res 11,3-11 (2001); Pease, A. et al., Proc Natl Acad Sci USA 91, 5022-5026(1994); Southern, E., Maskos, U. & Elder, J., Genomics 13, 1008-1017(1993); Wang, D. et al., Science 280, 1077-1082 (1998); Brown, P. &Botstein, D., Nat Genet 21, 33-37 (1999); Cargill, M. et al. 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J.,Clin Chem 43, 1749-1756 (1997); Armstrong, B., Stewart, M. & Mazumder,A., Cytometry 40, 102-108 (2000); Cai, H. et al., Genomics 69, 395(2000); Chen, J. et al., Genome Res 10, 549-557 (2000); Ye, F. et al.Hum Mutat 17, 305-316 (2001); Michael, K., Taylor, L., Schultz, S. &Walt, D., Anal Chem 70, 1242-1248 (1998); Steemers, F., Ferguson, J. &Walt, D., Nat Biotechnol 18, 91-94 (2000); Chan, W. & Nie, S., Science281, 2016-2018 (1998); Han, M., Gao, X., Su, J. & Nie, S., NatBiotechnol 19, 631-635 (2001); Griffin, T. & Smith, L., TrendsBiotechnol 18, 77-84 (2000); Jackson, P., Scholl, P. & Groopman, J., MolMed Today 6, 271-276 (2000); Haff, L. & Smirnov, I., Genome Res 7,378-388 (1997); Ross, P., Hall, L., Smirnov, I. & Haff, L., NatBiotechnol 16, 1347-1351 (1998); Bray, M., Boerwinkle, E. & Doris, P.Hum Mutat 17, 296-304 (2001); Sauer, S. et al., Nucleic Acids Res 28,E13 (2000); Sauer, S. et al., Nucleic Acids Res 28, E100 (2000); Sun,X., Ding, H., Hung, K. & Guo, B., Nucleic Acids Res 28, E68 (2000);Tang, K. et al., Proc Natl Acad Sci USA 91, 10016-10020 (1999); Li, J.et al., Electrophoresis 20, 1258-1265 (1999); Little, D., Braun, A.,O'Donnell, M. & Koster, H., Nat Med 3, 1413-1416 (1997); Little, D. etal. 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The following additional genotyping methods are also encompassed by thepresent invention: the methods described and/or claimed in U.S. Pat. No.6,458,540; and the methods described and/or claimed in U.S. Pat. No.6,440,707.

Example 10 Method of Limiting the Risk of an HIV-1 Protease Inhibitorfrom Causing Metabolic Abnormalities in a Patient by Measuring BasalResisting Levels

The inventors hypothesis that some HIV-1 protease inhibitors maysignificantly elevate human resistin plasma/serum levels in patients. Asdescribed herein, this effect is believed to be representative of HIV-1protease inhibitors that are also capable if inhibiting cathepsin D andcathepsin E. As a consequence of resistin being associated with obesityand insulin resistance, it is plausible to associate increased resistinplasma/serum levels both prior to or after administration of an HIV-1protease inhibitor with an increased susceptibility to a patient indeveloping HIV-1 protease inhibitor-dependent metabolic abnormalities orrelated disorder upon the administration of a HIV-1 protease inhibitor.Therefore, the present invention encompasses the use of assays designedto measure resistin plasma/serum levels prior or in in response to HIV-1protease inhibitor exposure in patients to identify patients that may beat risk of developing HIV-1 protease inhibitor-dependent metabolicabnormalities.

Resistin may be assayed from patient samples essentially as described byYagmor, et al., (Amer. J. Gastro., 101(6):1244 (2006)). Briefly, serumresistin concentrations can be determined using a quantitative sandwichenzyme immunoassay (ELISA), according to manufacturer's instructions(BioVendor, LLC, Candler, N.C., USA). Patient samples are stored at −80°C. at time of collection and remain freezed up to the time of resistinmeasurement. To ensure assay maintains a linear measurement, human serummay be diluted in a range from 1:2 to 1:8, as necessary.

Generally, though any method capable of measuring the plasma/serum levelof resistin would constitute a suitable method for identifying patientsat increased risk of developing HIV-1 protease inhibitor-dependentmetabolic abnormalities or related disorders. The lower the level ofresistin, the lower the likelihood that a patient will develop HIV-1protease inhibitor-dependent metabolic abnormalities.

Such an assay would also be useful for identifying levels of a givenHIV-1 protease inhibitor compound in vivo that has a lower likelihood ofinducing HIV-1 protease inhibitor-dependent metabolic abnormalities orrelated disorder in a patient. For example, it would be possible toidentify a dose of a HIV-1 protease inhibitor compound using the abovementioned assay that induces lower levels of resistin plasma/serumlevels, but that still has an acceptable level of efficacy.

The entire disclosure of each document cited (including patents, patentapplications, journal articles, abstracts, laboratory manuals, books, orother disclosures) in the Background of the Invention, DetailedDescription, and Examples is hereby incorporated herein by reference.Further, the hard copy of the Sequence Listing submitted herewith andthe corresponding computer readable form are both incorporated herein byreference in their entireties.

While this invention has been particularly shown and described withreferences to preferred embodiments thereof, it will be understood bythose skilled in the art that various changes in form and details may bemade therein without departing from the scope of the inventionencompassed by the appended claims.

It will be clear that the invention may be practiced otherwise than asparticularly described in the foregoing description and examples.Numerous modifications and variations of the present invention arepossible in light of the above teachings and, therefore, are within thescope of the appended claims.

The entire disclosure of each document cited (including patents, patentapplications, journal articles, abstracts, laboratory manuals, books, orother disclosures) in the Background of the Invention, DetailedDescription, and Examples is hereby incorporated herein by reference.Further, the hard copy of the Sequence Listing submitted herewith andthe corresponding computer readable form are both incorporated herein byreference in their entireties.

While this invention has been particularly shown and described withreferences to preferred embodiments thereof, it will be understood bythose skilled in the art that various changes in form and details may bemade therein without departing from the scope of the inventionencompassed by the appended claims.

1. A method of identifying an individual who may be at risk ofdeveloping HIV-1 protease inhibitor-dependent metabolic abnormalitiesupon administration of a HIV-1 protease inhibitor comprising the stepsof (a) obtaining a nucleic acid sample from an individual; and (b)determining whether said individual has a reference or variable alleleat nucleotide position 1398 of SEQ ID NO:1 or SEQ ID NO:2, wherein thepresence of a reference “C” allele at said nucleotide position isindicative of a decreased risk of developing HIV-1 proteaseinhibitor-dependent metabolic abnormalities in an individual receivingHIV-1 protease inhibitor therapy relative to an individual having thevariable “T” allele at said nucleotide position.
 2. The method accordingto claim 1, wherein said individual is heterozygous for the reference“C” allele.
 3. The method according to claim 1, wherein said individualis homozygous for the reference “C” allele.
 4. The method according toclaim 1, wherein said individual is heterozygous for the variable “T”allele.
 5. The method according to claim 1, wherein said individual ishomozygous for the variable “T” allele.
 6. The method according toclaims 2 or 4, wherein said individual has a decreased risk ofdeveloping HIV-1 protease inhibitor-dependent metabolic abnormalitiesrelative to an individual that is homozygous for the variable “T”allele.
 7. The method according to claim 3, wherein said individual hasa decreased risk of developing HIV-1 protease inhibitor-dependentmetabolic abnormalities relative to an individual that is heterozygousfor the reference “C” allele or variable “T” allele, and relative to anindividual that is homozygous for the variable “T” allele.
 8. The methodaccording to claim 5, wherein said individual has an increased risk ofdeveloping HIV-1 protease inhibitor-dependent metabolic abnormalitiesrelative to an individual that is heterozygous for the reference “C”allele or variable “T” allele, and relative to an individual that ishomozygous for the reference “C” allele.